advantages and limitat

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ELSEVIER Mutation Research 348 (1995) 153-162M u t a t i o n R e s e a r c h L e t t e rs

Advantages and l imi ta t ions of us ing f luorescence in s i tuhybrid izat ion for the detect ion of aneuplo idy in in terphase

human cel ls

David A. Eas tm ond * , M aik Schu le r, D.S . Rup aEnz'ironmental Toxicology Graduate Program Department of Entomology Unicersity of California Rit'erside, CA 92521, USA

Received 15 August 1995; revised 29 Sep tember 1995: accepted 27 Sep tembe r 1995

A b s t r a c t

Fluorescence in situ hybridization with chromosome-specific DNA probes is being increasingly utilized for thedetect ion of chromosome aberrations induced in vitro and in vivo by chemical and physical agents. Althoughpotentially a powerful technique, FISH studies for aneuploidy can be heavily influenced by cellular phcnomcna andhybridization 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 wouldexpect based upon frequencies seen in conventional mctaphase analyses. In this article, a number of the potentialpitfalls 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 oflymphocyte nuclei containing 3 and 4 chromosome copies are compared between mctaphase values obtained frompublished human population studies and interphasc values obtained from similar studies using FISH. It is concludedthat by using caution in the evaluation of slides, interphase studies using FISH to detect hyperdiploidy andpolyploidy can provide estimates of numerical alterations which closely reflect those seen during mctaphase analysisusing either FISH or conventional approaches. However, duc to the inability of interphase analysis to distinguishhyperdiploidy from polyploidy as well as other potential problems, frcqucncics of aneuploid nuclei obtained usingsingle 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 . I n t r o d u c t i o n

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

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

ing increasingly utilized in genotoxicity studies todetect chromosome aberrations induced in vitroand in vivo by chemical and physical agents. Overthe past several years, a variety of probes andlabeling strategies have been employed to detector characterize chromosomal alterations. For ex-ample, probes labeling the centromeric region of

0165-7992/95/$ 09.50 1995 Elsevier Science B.V. All rights reservedSSDI 01 65-7992(95 )0(1059-3


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154 D . A . E a s t m o n d e t a l . / M u t a t i o n R e s e a r c h 3 4 8 (1 9 9 5 ) 1 5 3 - 1 6 2

a l l o r n e a r l y a l l h u m a n o r m o u s e c h r o m o s o m e sh a v e b e e n u s e d t o c h a r a c t e r i z e t h e o r i g i n o f m i -c r o n u c l e i o c c u r r i n g s p o n t a n e o u s l y o r f o l l o wi n ge x p o s u r e t o v a r i o u s c h e m i c a l a g e n t s ( B e c k e r e ta l . , 1990; Mil ler e t a l . , 1991; Chen e t a l , 1994;

Ti t e n k o - Ho l l a n d e t a l . , 1 9 9 4 ) . O t h e r p r o b e s wh i c hl a b el t h e l e n g t h o f a sp e c i f ic c h r o mo s o m e , c o m-m o n l y k n o w n a s w h o l e c h r o m o s o m e p r o b e s o rp a i n t i n g p r o b e s , h a v e b e e n s h o wn t o b e v a l u a b l ei n d e t e c t i n g a n d q u a n t i f y i n g s t r u c t u r a l c h r o mo s o -ma l a b e r r a t i o n s , p a r t i c u l a r l y t r a n s l o c a t i o n s a n dd i c e n t r i c s o c c u r r i n g b e t w e e n n o n h o m o l o g o u sc h r o m o s o m e s i n m e t a p h a s e p r e p a r a t i o n s ( N a t a -ra jan e t a l . , 1992; "Fucker e t a l . , 1993). Ad di t ion -a l l y, p r o b e s wh i c h t a rg e t c e n t r o me r i c o r p c r i c e n -t r o me r i c s a t e l l i t e s e q u e n c e s h a v e b e e n u s e d t od e t e c t a n e u p l o i d y, i . e . n u m e r i c a l c h r o m o s o m ec h a n g c s , a n d m o r e r e c e n t ly c h r o m o s o m a l b r e a k -a g e a n d e x c h a n g e s , i n d u c e d b y c h e mi c a l a n dp h y s i c a l a g e n t s i n me t a p h a s e a n d i n i n t e r p h a s ece i l s (E as t mo nd and Pinke l , 1990 ; De S a r io e t a l .,199( I; Ru pa e t a l ., 1994). Fo r a nu m be r o f yea r s ,w e h a v e u s e d c e n t r o m e r i c D N A p r o b e s t o d e t e c ta n e u p l o i d y a n d o t h e r c h r o m o s o m e a l t e r a t i o n s i nin te r pha se ce l ls . The ob jec t ive o f th i s a r t i c l e i s tod i s cu s s s o me o f th e s t r e n g t h s a n d l i m i t a t io n s o fi n t e r p h a s e F I SH a n a l y s i s t o a i d r e s e a r c h e r s a st h e y b e g i n p e r f o r mi n g F I SH s t u d i e s a n d t o a s s i s to t h e r s i n v o l v e d i n t h e i n t e r p r e t a t i o n o f F I SHr e s u lt s . A l t h o u g h mo s t o f t h e s e c o m me n t s a r ea p p l i c a b l e t o ma n y t y p e s o f F I SH s t u d i e s , t h ep r i ma r y f o c u s o f th i s a r t i c le w i ll b e o n t h e u s e o fc h r o m o s o m e - s p e c i f i c c e n t r o m e r i c p r o b e s t o d e -t e c t a n e u p l o i d y i n s o ma t i c c e i l s .

Th e u s e o f F I SH f o r c y t o g e n e t i c a n a ly s i s isg a i n i n g i n c r e a s i n g a c c e p t a n c e p r i ma r i l y d u e t o i t se a s e o f u s e a n d t h e s p e e d o f a n a ly s i s. Th o u s a n d sof ce l ls can be e va lu a ted in a r e l a t ive ly shor tp e r i o d o f t i me i n c r e a s i n g s a mp l e s i z e s a n d e n -

h a n c i n g s t a t i s t i c a l p o we r. Th i s i n c r e a s e s a s s a ysens i t iv i ty and a l lows sma l le r inc reases in induceda l t e r a t i o n s t o b e d e t e c t e d . An a d d i t i o n a l a n dp o s s ib l y mo r e i m p o r t a n t r e a s o n i s t h a t i n t e r p h a s ea n a ly s i s c a n b e p e r f o r m e d o n r a r e l y d i v id i n g o rn o n d i v i d i n g c e l l s a l l o wi n g c y t o g e n e t i c i n f o r ma -t i o n t o b e o b t a i n e d f r o m t i s s u e s wh i c h p r e v i o u s l yh a v e n o t b e e n a m e n a b l e t o c y t o g e n e t i c an a l y si s(Sandberg e t a l . , 1988 ; Teyss le r, 1988) . Al ready,

i n t e r p h a s e a n a ly s i s h a s p r o v i d e d v a l u a b l e i n f o r -m a t i o n o n a n e u p l o i d y i n h u m a n s p e r m , u n s t i m u -l a t e d l y m p h o c y t e s , b u c c a l mu c o s a l c e l ls a n du r o t h e l i a l c e ll s ( Ea s t m o n d a n d P i n k e l , 1 99 0;M oo re e t a l ., 1993; Bis cho ff e t a l . , 1994) .

2. Technica l l imi ta t ions and poten t ia l p i t fa l l s inF I SH a na ly s i s

Al o n g wi t h t h e a d v a n t a g e s o f b e i n g a b le t oa n a l y z e i n t e r p h a s e c e l l s c o me s o n e c l e a r d i s a d -v a n t a g e - t h e i n a b i li t y t o d i r e c tl y s e e t h e c h r o m o -s o m e a n d c h r o m o s o m a l r e g i o n s t a r g e t e d b y t hep r o b e . Co n s e q u e n t l y, o n l y a l t e r a t i o n s a f f e c t i n gt h e l a b e l e d r e g i o n c a n b e d e t e c t e d . I n a d d i t i o n ,h y p e r d i p i o i d f r e q u e n c i e s f o r i n d i v i d u a l c h r o mo -s o me s a r e f r e q u e n t l y r e p o r t e d i n t h e l i t e r a t u r ewh i c h a r e s u b s t a n t i a l l y h i g h e r t h a n o n e wo u l de x p e c t b a s e d u p o n t h e r e s u l t s o f c o n v e n t i o n a lme t a p h a s e a n a l y s e s ( K i b b e l a a r e t a l ., 1 9 93 ; LeBeau , 1993 ; Her r ing ton e t a l . , 1995) . Th i s ind i -c a t e s t h a t c e r t a i n t y p e s o f h y b r i d i z a t i o n p a t t e r n so r t e c h n i c a l p r o b l e ms ma y r e s u l t i n e u p l o i d c e l l sb e i n g c l a s s i f i e d a s a n e u p l o i d a n d t o a l e s s o r e x -t e n t a n e u p l o i d c e l l s b e i n g c h a r a c t e r i z e d a s e u -p lo id .

Ty p i c a l l y i n t e r p h a s e F I S H s t u d i e s u t i l iz e o n eo r t wo p r o b e s s i mu l t a n e o u s l y t o q u a n t i f y v a r i a -t i o n s i n c h r o m o s o m e n u m b e r , w h i c h m e a n s o n l ya l t e r a t i o n s a f f e c t i n g a l im i t e d p o r t i o n o f t h eg e n o me c a n b e d e t e c t e d . A l t h o u g h t h i s r e s u l t s i nd e c r e a s e d s e n s i t i v i t y, t h e g r e a t l y i n c r e a s e d s p e e do f i n t e r p h a s e a n a l y si s m i n i mi z e s t h i s p r o b l e m. I ns tud ie s o f somat ic ce l l s , nuc le i exh ib i t ing 3 o rmo r e h y b r i d i z a t i o n r e g i o n s a r e c l a s s i f i e d a s h y -p e r d i p l o i d w h e r e a s t h o s e w i t h 0 o r 1 h y b r i d i z a -t i o n r e g i o n a r e c a l l e d h y p o d i p i o i d . We u s e t h ewo r d ' h y p e r d i p l o i d ' l i t e r a l l y, me a n i n g g r e a t e r

than d ip lo id so i t i nc ludes ce l l s wh ich a re t r i som ica n d t e t r a s o m i c f o r t h e l a b e l e d c h r o m o s o m e a swe l l a s po lyp io id ce l l s (4N, 8N) . The inc lus ion o fp o l y p l o i d c e i l s i s i mp o r t a n t wh e n c o mp a r i n g i n -t e r p h a s e w i t h me t a p h a s e r e s u l t s . Ce l l s c l a s s i f i e das hypo d ip lo id inc lu de ce l l s wh ich have lo s t 1 o rmo r e c h r o mo s o m e s a s we ll a s c e l ls wh i c h l a c ko n e s i g n a l d u e t o h y b r i d i z a t i o n a r t i f a c t s o r n u -c l e a r o rg a n i z a t i o n i n i n t e r p h a s e c e l ls . Ex a m p l e s


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D.A. East mo nd et a l. /M ut at io n Research 348 (1995) 153-1 62 155

o f p r o b l e m s t h a t m a y r e s u l t in a n i n a c c u r a t ei n t e r p r e t a t i o n o f F I S H r e s u lt s a r e s u m m a r i z e d i nTa b l e 1. S o m e c o m m o n f c a t u r e s o f t h c s c p a t t e r n so r p r o b l e ms wi l l b e d e s c r i b e d i n mo r e d e t a i lb e l o w.

Oc c a s i o n a l l y f o l l o wi n g F I SH, c e l l s wh i c h c o n -t a i n 3 h y b r i d i z a t i o n s i g n a l s a r e o b s e r v e d i n c o n -t r o l o r u n t r e a t e d c u l t u r e s . W h e n t wo s i g n a l s a p -pea r a s a doub le t [ see Fig . l a ; hav ing a ve rys i mi l a r a p p e a r a n c e w i t h o n l y a s ma ll s p a c e s e p a -r a t i n g t h e a d j a c e n t p r o b e s ; b o t h b e i n g s ma l l e rt h a n t h e t h i r d s i g n a l wh i c h i s mo r e n o r ma l i n s i z ef o r n u c l e i o f t h i s d i a me t e r ] , t h e d o u b l e t s i g n a l sa r e s c o r e d a s o n e h y b r i d i z a t i o n r e g i o n a n d a r et h o u g h t p o s s ib l y t o b e e i t h e r c e l ls i n t h e G 2 s t a g eo f t h e c e ll cy c l e o r t h e r e s u l t o f p r e m a t u r e c h r o -ma t i d s e p a r a t i o n d u r i n g f i x a t i o n o r d r y i ng . [ Th ea p p e a r a n c e o f o n ly o n e d o u b l e t is n o t s u rp r is i n ga s i t wo u l d b e h i g h l y u n l i k e l y t h a t b o t h c h r o mo -s o me s wo u l d l a n d o n t h e s l i d e i n a n o r i e n t a t i o nt h a t w o u l d p e r m i t s e p a r a t i o n b e t w e e n t h e h y -b r i d i z a t i o n r e g i o n s o n b o t h p a i r s o f s i s t e r c h r o -ma t i d s t o b e s e e n . ] I n a d d i t i o n ,alpha sa te l l i t eD N A w h i c h s p a n s t h e p r i m a r y c o n s t r i c ti o n w h i chma y a l s o r e s u l t i n t h e s e d o u b l e t - t y p e s i g n a l s( H e r r i n g t o n e t a l ., 1 9 9 5) . O t h e r c e l l s c o n t a i n i n g 3h y b r i d i z a t i o n r e g i o n s a r e o c c a s i o n a l l y s e e n i nw h i c h a th i n t h r e a d o f D N A c o n n e c t s t w o o f t h eh y b r i d i z a t i o n r e g i o n s . Th e s e c o n n e c t e d h y b r i d i z a -

t i o n r e g i o n s c a n b e w i d e l y s e p a r a t e d w i t h i n t h en u c l e u s ( s e e F i g . l b ) . Th i s p a t t e r n ma y r e p r e s e n tc e l l s i n wh i c h t h e t a rg e t r e g i o n i s d e c o n d e n s e dd u r i n g DNA r e p l i c a t i o n o r r e p a i r r e s u l t i n g i nh y b r i d i z a t i o n r e g i o n s t h a t h a v e mi g r a t e d a wa y

f r o m e a c h o t h e r d u r i n g h y p o t o n i c t re a t m e n t , f ix a -t i o n o r d r y i n g o n t h e s l id e . I n o u r l a b o r a t o r y, wes c o r e w i t h a c o n s e r v a t i v e b i a s s o t h a t n u c l e i c o n -t a i n i n g t h e a b o v e - d e s c r i b e d h y b r i d i z a t i o n p a t -t e r n s a r e s c o r e d a s e u p l o i d .

T h e a p p e a r a n c e o f 3 fl u o r e s c e n t s po t s c a n a l sor e f le c t c h r o m o s o m e b r e a k a g e o r e x c h a n g e s . I ns c o r i n g c e ll s f o ll o wi n g t r e a t m e n t w i t h c l a s t o g e n i ca g e n t s , we s a w a h i g h f r e q u e n c y o f n u c l e i c o n t a i n -i n g 3 h y b r i d i z a t i o n r e g i o n s wh i c h a r e a l mo s t c e r -t a i n ly th e r e s u l t o f b r e a k a g e w i t h i n t h e c h r o mo -s o m e r e g i o n t a r g e t e d b y t h e D N A p r o b e r a t h e rt h a n t h e p r e s e n c e o f t h r e e i n d i v i d u a l c h r o m o -s o m e s . T h i s p r o b l e m i s p r o n o u n c e d w h e n w o r k -ing wi th c la s s ica l sa te l l i t e p rob es wh ich t a rge t thec e n t r o m e r i c h e te r o c h r o m a t i n o f h u m a n c h r o m o -som es 1 , 9 , and 16 , r eg ions wh ich a re p ro ne tob r e a k a g e b y c h e m i c a l a n d p h y s ic a l a g e n t s(Brogger, 1977 ; Rupa e t a l . , 1995) . Th i s can com-p l i c a t e t h e i n t e r p r e t a t i o n o f s tu d i e s o n a n e u -p l o i d y i n t r e a t e d a n d u n t r e a t e d c e l l s , p a r t i c u l a r l yw h e n t e s t i n g c l a s t o g c n i c a g e n t s ( E a s t m o n d a n dPinke l , 1990 ; Eas tmond e t a l . , 1994) . An ana lo -g o u s s i t u a t i o n i s s e e n wh e n u s i n g p r o b e s t h a t

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

t lybr id izat ion patterns that m ight be incorrectly in terpreted as hyperd ip lo idBreakage or exchanges affecting the chromosomal region targeted by the probeCells in th e G 2 stage o f the cell cyclePremature chromatid separationSatellite DNA s panning the primary constriction (e.g. due to pericentri c inversion)Cells in S phase with deconde nsed DNA co nnecting condensed satellite regionsCells undergoing DNA repair with decondensed DNA connecting condensed satellite regionsNonspecific binding of probe or cross hybridization with satellite regions on othe r chrom osomesFluorescen t debris, particularly combin ed with small hybridization signals

t tybr id izat ion patterns that m ight be incorrectly in terpreted as hypodip lo idStrong count erstain that o bscures small or weak hybridization signalsInefficient filter for detecting fluor ochrome of interestWeakening or loss of signals due to high stringency washesPoor hybridization due to poor quality of probe or inefficient probe pe netra tion into nucleusOverlapping of regions hybridizing to prob eNuclei from do nors with highly polymorphic satellite regionsNuclear organization in unstimulated and stimulated interphase cells


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15 6 D.A. Ea.$tmond et al. / Muta tion Research 3 48 (1995) 15 3-1 62

t a r g e t t h e b r e a k a g e - p r o n e m o u s e p c r i c e n t r i c h e t -e r o c h r o m a t i n ( Ch e n e t a l. , 1 9 94 ).

To d i s t i n g u i s h b r e a k a g e f r o m n u m e r i c a lc h a n g e s , we h a v e r e c e n t l y d e v e l o p e d a mu l t i c o l o rF I S H a p p r o a c h t o d e t e c t n u c l e i w i t h mu l t i p l e

h y b r i d i z a t i o n r e g i o n s f o r m e d f r o m b r e a k a g e

a

O

i ' I[ , , ~ / ,

T ' !I

.ZZ - _ ~ . ~ - " 3

Fi g . 2 . t t yb r i d i z a t i on s t r a t e gy u s i ng two a d j a c e n t o r t a nd e mD N A p r o b e s t o d e t ec t c h r o m o s o m e b r e a k a g e a n d h y p e r-d i p lo i d y i n m e t a p h a s e a n d i n t e r p h a s e c e i l s. [ F r o m R u p a e t a l .( 1 9 95 ) C a n c e r R e s . 5 5 , 6 4 0 - 6 4 5 , r e p r i n t e d w i t h p e r m i s s i o n o ft h e A m e r i c a n A s s o c i a t i o n f o r C a n c e r R e s e a r c h . ]

F i g. 1. l n t e r p h a s e h u m a n l y m p h o c y t e s f o ll o w i ng f l u o r e s c e n c ei n s i t u h yb r i d i z a t i on wi t h a c l a s s i c a l s a t e l l i t e p r ob e t o c h r om o-

s om e 9 a nd s t a i n i n g wi t h p r op i d i um i od i d e , a ) In t he l a rge rnuc l e u s , t h r e e h yb r i d i z a t i o n r e g i ons a r e v i s i b l e : t wo o f t h e s ea r e a d j a c e n t a nd s m a l l e r i n s i z e i l l u s t r a t i n g t he doub l e t p a t -t e r n d e s c r i b e d i n t he t e x t . I n t he s m a l l e r nuc l e us , o n l y onehy b r i d i z a t i o n s i g n a l i s v i s i b le . Ba s e d on t he nu c l e us a ndhyb r i d i z a t i o n s i z e s , t h i s s i gna l i s l i ke l y c om p o s e d o f t he s upe r -i m p o s e d s i g n a l s o f t w o c h r o m o s o m e s , b ) A n u c l e u s c o n t a i n i n g3 h y b r i d i z a t i o n r e g i o ns wi t h a t h i n hyb r i d i z e d b r i dg e c on n e c t -i n g t w o o f t he h y b r i d i z at i o n r e g i o n s . C o n n e c t e d h y b r i d i z a t io nr e g i o n s s u c h a s t h i s a r e s c o r e d a s o n l y one c op y o f t he l a be l e dc h r o m o s o m e .

(Eas tmond e t a l . , 1994 ; Rupa e t a l . , 1995) . Thebas i s o f th i s new F ISH assay is i l lu s t r a ted in Fig .2 . Th i s a p p r o a c h a l s o al l ows t h e c y t o g e n e t i c i s t t omo r e c o n f i d e n t l y a n d a c c u r a t e l y c l a ss i f y c e i ls a sa n c u p l o i d . I n t h i s a p p l i c a t i o n , mu l t i c o l o r F I SH i su s e d t o l a b e l t wo a d j a c c n t ( o r t a n d e m) r e g i o n s o nc h r o mo s o m e 1, 9 o r 1 6. Th e p e r i c e n t r i c h e t e -r o c h r o m a t i c r e g i o n wh i c h is l a rg e a n d p r o n e t ob r e a k a g e b y c h e mi c a l a n d p h y s i ca l a g e n t s i s l a -be l ed by a Tex as r ed "~', Rh od am ine o r C y-3 c las s i -c a l s a t e l li t e p r o b e . An a d j a c e n t c e n t r o m e r i c r e -g i o n wh i c h i s s o me wh a t s m a l l e r a n d mu c h l e s sp r o n e t o b r e a k a g e i s s t a i n e d w i t h a g r e e n f l u o r e s -c e i n - l a b e l e d alpha s a t e l l i t e p r o b e . Th e p r e s e n c e

o f a n i n t e r p h a s e n u c l e u s c o n t a in i n g t h r e ealphasa te l l i t e p robes ad jacen t to th ree c la s s ica l sa te l -l i t e p r o b e s i n d i c a t e s a n u c l e u s wh i c h h a s 3 c o p i e so f t he c h r o m o s o m e o f i n t er e s t. H o w e v e r i f as i mi l a r i n t e r p h a s e n u c l e u s c o n t a i n s o n l y t woal-pha s a t e l li t e p r o b e s a d j a c e n t t o t wo o f t h e t h r e ec la s s ica l sa te l l i t e p robes , th i s ind ica te s tha tb r e a k a g e h a s o c c u r r e d w i t h in t h e c h r o m o s o m a lr e g i o n t a rg e t e d b y o n e o f t h e c l a s s ic a l s a t e l li t e


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D.A. Eastmond et al. /Mutation Research 348 (1995) 153-162 15 7

p r o b e s . A l t e r n a t i v e l y, a w i d e s e p a r a t i o n b e t w e e nt h e r e g i o n s l a b e l e d b y t h ealpha and c la s s ica ls a t e l l i t e p r o b e s c a n a l s o i n d i c a t e t h a t c h r o mo s o -m a l b r e a k a g e h a s o c c u r r e d .

C e l l u l a r p h e n o m e n a o r h y b r i d i z a t i o n a r t i f a c t s

c a n a l s o i n f l u e n c e t h e f r e q u e n c y o f n u c le i wh i c hc o n t a i n 0 o r 1 h y b r i d i z a t i o n r e g i o n a n d r e s u l t i ne l e v a t e d f r e q u e n c i e s o f d i p l o i d c e l l s b e i n g c l a s s i -f i e d a s h y p o d i p l o i d . Fo r e x a mp l e , i f t wo l a b e l e dc h r o m o s o m e s l i e i m m e d i a t e l y a d j a c e n t o r o n t o po f o n e a n o t h e r, t h e h y b r i d i z a t i o n s i g n a l s w i ll o f_t e n b e s u p e r i m p o s e d a n d a p p e a r a s o n e s i g n a l( F i g . l a ) . Th e o c c u r r e n c e o f t h is e v e n t a p p e a r s t ob e h i g h ly i n f l u e n c e d b y t h e s i z e o f t h e n u c l e u sa n d t h e r e g i o n t a rg e t e d b y t h e p r o b e , b y n u c l e a ro r g a n i z a t i o n w h i c h d i f f e r s f o r e x a m p l e , b e t w e e ns t i m u l a te d a n d u n s t i m u l a t e d l y m p h o c y t c s a n dp o s s i b l y b y t h e u s e o f h y p o t o n i c t r e a t me n t s t oswe l l the nuc le i (Schmid e t a l . , 1983 ; Eas tmondand Pinke l , 1990 ; Fc rg uson and W ard , 1992 ; Lcwise t a l ., 1993). Idea l ly, on e w ou ld l ike to use the

s ma l l e s t p r o b e p o s s i b l e t o mi n i mi z e t h i s p r o b l e m.Ho w e v e r w i t h p r o b e s t o s ma l l r e g i o n s , s c o r i n gb e c o me s mo r e d i f f i c u l t a s t h e v i s i b i l i t y o f h y -b r i d i z a t i o n s i g n a l s i n i n t e r p h a s e c e l l s t e n d s t od e c r e a s e a n d t h e s i g n a l s b c c o mc h a r d e r t o d i s t i n -

gu i sh f rom f luo rescen t a r t i f ac t s . In add i t ion , ce i l sma y b c i n c o r r e c t l y i d e n t i f i e d a s h y p o d i p l o i d d u ct o s u b - o p t i ma l h y b r i d i z a t i o n o r mi c r o s c o p e d e t e c -t i o n c o n d i t i o n s . Th e u s e o f a n u c l e a r c o u n t c r s t a i na t a n e x c e s s i v e c o n c e n t r a t i o n c a n o b s c u r e s ma l lh y b r i d i z a t i o n s i g n a l s a s c a n t h c u s e o f i n e f f i c i e n tmi c r o s c o p i c f i l t e r s a n d wa s h i n g s l i d e s a t e x c e s -s iv e ly h ig h s t r i n g e n c y. Fo r e x a m p l e , t h e c f f c c t s o fd i f f e r e n t s l i d e p r e p a r a t i o n c o n d i t i o n s o n u n -t r e a t e d a n d d i e t h y l s t i l b e s t r o l - t r e a t e d c e l l s i ss h o wn i n Ta b l e 2 . I n t h i s e x a mp l e , t h e o b s e r v e dh y b r i d i z a t i o n f r e q u e n c i e s h a v e b e e n s i g n i f i c a n t l yi n f l u e n c e d b y t h e p r o b e u s e d , t h e s t r i n g e n c y o ft h e wa s h , th e s t r e n g t h o f th e c o u n t e r s t a i n a n d t h ec h e m i c a l t r e a t m e n t . F u r t h e r m o r e , p e n e t r a t i o np r o b l e ms d u e t o r e s i d u a l c e l l me mb r a n e s o r h i g h

Ta b l e 2I n f l u e n c e o f h y b r i d i z a t io n a n d s t a i n i n g c o n d i t i o n s o n t h e f r e q u e n c i e s o f h y p o - a n d h y p e r d i p l o i d y i n u n t r e a a n d d i e t h y l s t i lb e s t r o l( D E S ) - t r e a t e d c u l t u r e d h u m a n l y m p h o c y t e s d e t e r m i n e d u s i n g f l u o r e s c e n c e i n s i tu h y b r i d i za t i o n w i t h a nalpha a n d a c l a s s i c a ls a t e ll i te p r o b e f o r c h r o m o s o m e 1

C o m p o u n d D N A Wa s h i n g P I C o n c . " N u m b e r o f h y b r id i z at i on H y pe r d ip l o id D e s c r ip t i onp r o b e c o n d i ti o n s ( p . g / m l ) s i g n a l s / 1 0 0 0 c el ls ( % )

0 1 2 3 4

0 . 1 % D M S O alpha I 60% f o r m a m i d e / 0 . 22 X S S C ; 4 5 C

c l a ss i c al 1 5 0 % f o r m a m i d e / 0 . 22 X S S C : 4 5 C

2

6 0% f o r m a m i d e / 0 .22 X S S C ; 4 5 C

2

D E S 2 0 / . t M a l p h a 1 6 0 ~ : f o r m a m i d e / 0 .22 X S S C ; 4 5 C

c l a ss i c al 1 5 ( 1 % f o r m a m i d e / ( }.22 X S S C ; 4 5 C

2

6 0 % f o r m a m i d e / 0 .22XSSC; 4 5 ( ?

2

(I 57 940 2 1 0.3

6 92 899 3 0 0 .3

9 133 855 2 1 0.3

21 153 822 3 1 (1.4

28 155 814 3 0 0.3

3 63 848 15 72 8 .7

2 76 837 35 51) 8.5

12 115 799 31 43 7.4

15 134 776 31 44 7.5

32 143 794 12 19 3.1

Opt im a l hybr id i za t i on cond i ti ons

O p t i m a l h y b r i d i z a t i o n c o n d i t i o n s

E x c e s s iv e c o u n t e r s t a i nc o n c c n t r a t i o nExc e s s i ve l y s t r i nge n tw a s h i n g c o n d i t i o n sE x c e s s iv e c o u n t e r s t a i n a n da n d c o n d i t i o n s

O p t i m a l h y b r i d i z a t io n c o n d i t i o n s

O p t i m a l h y b r i d i z a t io n c o n d i t i o n s

E x c e s s iv e c o u n t e r s t a i nc o n c e n t r a t i o nExc e s s i ve l y s t r i nge n tw a s h i n g c o n d i t i o n sE x c e s s i ve c o u n t e r s t a i n a n dw a s h i n g c o n d i t i o n s

a PI , p rop id ium iod ide .


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158 D . A . E a s t m o n d e t a l. / M u t a t i o n R e s e a r c h 3 4 8 ( 1 9 9 5 ) 1 5 3 - 1 6 2

n u c lea r co m p ac t io n can a l so r e su l t i n w eak h y -br id izat ion s ignals which can resu l t in e levatedf req u en c ies o f ce i ls ev a lu a ted as h y p o d ip lo id .

S u b -o p t im a l h y b r id i za t io n co n d i t io n s can a l soresu l t in hyperd ip lo id cel ls being scored as d ip lo id .

As i l lus t ra ted fo r the DES-t reated cei l s in Tab le2 us ing the c lass ical sa te l l i te p robe fo r ch romo-so m e 1 , t h e f r eq u en cy o f h y p erd ip lo id n u c le i d e -c l in ed b y g rea te r t h an 5 0 % ( f ro m 8 .5 % to 3 .1 % )u n d er co n d i t io n s o f ex cess iv e co u n te r s t a in an delevated s t r ingency wash ing . I t i s in teres t ing ton o te th a t a 5 0 % d ec rease f ro m , fo r ex am p le ,0 .4 % to 0 .2 % w o u ld m o s t l ik e ly n o t b e n o t i ced inscor ing 1000 to 20( ,ud un t rea ted ceil s . F ur the r-m o re , o v e r l ap p in g o f h y b r id i za tio n r eg io n s , p ro b -ab ly resu l ts in nucle i con ta in ing 3 o r 4 hybr id iza-t io n r eg io n s b e in g sco red as co n ta in in g 2 an d 3hybr id izat ion reg ions , respect ively. Th is can beseen b y co m p ar in g th e h y p erd ip lo id f r eq u en c iesin Tab le 2 o b ta in ed fo r t h ealpha sate l l i te p robeand the larger c lass ical sa te l l i te p robe which ex-h ib i ts in terp hase associa t ions (Schmid e t a l ., 1983;F crg u so n an d W ard , 1 9 9 2 ). In ad d i t io n fo l lo w in gnondis junct ion (o r endoredup l ica t ion) , i t i s l ikelyth a t t h e cen t ro m er ic r eg io n s o f s is t e r ch ro m at id swhich have fa i led to d is jo in wi l l be located inclose physical p rox im i ty in the s ubs equ en t in ter-phase cell . As a result , i t is possible that te-t r ap lo id o r en d o red u p l i ca t ed ce il s will f r eq u en t lyexh ib i t 2 o r 3 larger hybr id izat ion reg ions ra therthan an expected 4 s ignals .

3 . U s e o f F IS H fo r s tu d i e s o f a n e u p lo id y

As a resu l t o f these po ten t ia l l imi ta t ions , web e l i ev e th a t cau t io n sh o u ld b e u sed in sco r in gan d in th e in t e rp re t a t io n o f th e r e su l t s o f F IS Hanalyses . Based on our exper ience as wel l as the

d a ta p resen ted in Tab le 2 , p a r t i cu la r cau t io nsh o u ld b e ex e rc i sed in in t e rp re t in g F IS H s tu d iesw h en th e co m b in ed f r eq u en c ies o f n u c le i w i th 0and 1 hyb r id izat ion s ignals in un t re ated cel ls ex -ceed 1 5 % . H o w ev er, i n m o s t s i t u a t io n s w i th w e l ld es ig n ed ex p er im en t s , a co m p ar i so n o f th e t r ea t edce l l s o r ex p o sed h u m an s o r an im a l s w i th ap p ro -pr ia te con tro ls p rov ides substan t ia l ev idence thatth e ag en t i n d u ces a l t e ra t io n s in ch ro m o so m e

n u m b er. U s in g th i s ap p ro ach , cen t ro m er ic D N Ap ro b es h av e b een sh o w n to b e a v a lu ab le to o l fo rth e id en t i f i ca t io n o f an eu p lo id y in ce l l s ex p o sedto chemical and physical agen ts in v i t ro as wel l asfo r ch em ica l ly ex p o sed h u m an p o p u la t io n s . In

v i t ro s tu d ies h av e d em o n s t ra t ed th a t h y b r id i za -t io n s t r a teg ies can e f f ec tiv e ly d e tec t an eu p lo id yinduced by a var ie ty o f agen ts such as co lch ic ine ,v incr is t inc su l fa te , d ie thy ls t i lbes t ro l , ch lo ra l hy-d r a t e , B e n o m y l , g r i s e o f u l v i n , h y d r o q u i n o n e ,1 , 2 , 4 - b e n z e n e t r i o l , v a n a d i u m p e n t o x i d e a n dso d iu m a r sen i t e (R a im o n d i e t a l ., 1 9 8 9 ; E as t -mo nd and P inkel , 19 90 ; De Sar io e t a l. , 1990 ;Vag narel l i e t a i ., 1 990 ; Zh an g e t a l ., 1994 ; East -mo nd e t a l ., 19 94 ; Ram irez e t a l . , 1995) . In add i-t ion , th is approach is s tar t ing to be app l ied toce l l s o b ta in ed f ro m h u m an p o p u la t io n s . A l th o u g hm an y o f th e s tu d ies to d a te h av e o n ly b een r e -cen t ly pub l ished as abs t racts o r are no t yet pub-l i sh ed , i n t e rp h ase s tu d ies u s in g F IS H w i th cen -t ro m er ic p ro b es h av e d e tec t ed e l ev a ted f r eq u en -c ies o f ch ro m o so m al a l t e ra t io n s in cu l tu red lym -p h o cy tes o b ta in ed f ro m p es t i c id e -ex p o sed w o rk -ers (Ru pa e t a l ., 1995) , c igare t te sm okers (East -m o n d , R u p a , H a s e g a w a a n d T h o m p s o n , u n p u b -l i sh ed r e su l t s ) an d b en zen e-ex p o sed w o rk er s(Smith e t a l . , 1 995 ) as wel l as in the b uccalm u co sa l ce ll s o f b e te l q u id ch ew ers (R u p a an dE as tm o n d , u n p u b l i sh ed r e su l t s ) an d th e sp e rm o fp a t i en t s r ece iv in g ch em o th e rap y (R o b b in s e t a l . ,1994).

A seco n d p o ten t i a l ad v an tag e o f F IS H an a ly -ses i s to p rov ide es t imates o f aneup lo idy occur-r ing in var ious nond iv id ing cel l s . Th is wi l l a l lowth e f r eq u en c ies o f an eu p lo id y to b e d e te rm in edfor ce l l types which do no t d iv ide read i ly incu l tu re . Addi t ional ly, th is wi l l p rov ide es t imateso f an eu p lo id y th a t a re n o t i n f lu en ced b y ce l lcu l tu re co n d i t io n s o r m i to g en ic s t im u la t io n , an d

m ay th e re fo re b e m o re r ep resen ta t iv e o f th e f r e -q u en cy o f an eu p lo id y in viv o.

4 . C o m p a r i s o n o f F IS H a n d c o n v e n t i o n a lme ta p h a s e a n a ly s i s

E s t im a tes o f an eu p lo id y in h u m an p e r ip h e ra lb lo o d ly m p h o cy tes v a ry w id e ly b e in g in f lu en ced


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D.A. Eastmond et al./Mutation Research 348 (19951 153-162 159

by the age and sex of the donor (Fitzgerald andMcEwan , 1977; Galloway and Buc kton, 1978), thescoring criteria (Verschaev e et al., 1978) and pos-sibly the length of cell culture as well as thedensity of metapha ses o n the slide. In spite of

these var iab les , a comparison of the aneuplo idyf requenc ies ob t a ined u s ing conven t iona l me thodswith those found us ing FISH techniques can pro-vide an estimate of the accuracy of FISH forin terphase analys is of cu l tured lymphocytes andprovide insight s of the value of FISH for othercell types. Since hypodiploidy is difficult to esti-mate with accuracy by both convent ion al andFISH approaches (due to chromosome loss dur-ing metaphase prepara t ions or the over lap andfusion of hybridization signals in interphase cells),this comparison will focus on the frequencies ofhyperdip lo idy and polyplo idy observed us ing thetwo types of analyses. For the metaphase analy-ses, 17 separ ate studies were locate d which con-ta ined a neuplo id y data for 43-56 hr lymphocytecul tures . A summary of these exper imenta l re-sults is listed in Table 3. Sixteen studies had

useful values for hyperdiploidy and 7 for poly-ploidy.

Frequenc ies of hyperdip lo idy ranged from 0 to4.17% with a mean of 0.73% (standard deviationof 1.1%). The mean for the polyploidy frequen-

cies was 0.17% (standard deviation of 0.19%;range 0-0 .56%). In comparison , we have con-ducted 2 popula t ion s tudies us ing FISH with cen-tromeric probes on in terphase lymphocytes s t imu-lated with PHA and cultured for 48 hr. Using thetandem probes for chromosome 1 , hybrid iza t ionfrequencies were de te rmine d for 19 indiv idualsserving as controls for a study of pesticide expo-sure (Rupa ct al. , 1995). The average frequencyof nucle i conta in in g 3 and 4 copies of chromo-some 1 was 0.17% (st anda rd devi ation 0.09%;range 0-0 .3%) . [Note : This inc ludes both t ruehyperdiploid and polyploid nuclei]. In a secondunpublished study, FISH with a single classicalsatellite probe for chro moso me 1 was perf orme don 57 nonsm oking males and females who com-prised the controls for a study of cigarette smok-ing . The average f requency of nucle i conta i n ing 3

Table 3Summary of aneuploidy and polyploidydata from metaphasc analysis of 43-56 h lymphocytecultures reported in the literature

Gender No Age Cells/person Total Termination Hypodiploid Hyperdiploid Polyploid Relerence(approximate) no. of time (%) (%.) (%)cells

F 17 19-74 100 1700 43-48 4.8 I).94 0.12 Hirai, 1970NR ~ 16 NR 96 1534 51 6.1 0 h NR Li nlecki t a l., 1971M + F ~ 67 18-87 100 6595 48 5.1 I).59 NR Fitzgerald and McEwan, 1977M + F 166 0-89 50 6401 48 4.2 I).58 NR Ga llowa y nd Buckton, 1978 cM 10 34 100 1000 52 1.4 0.1 0 Popescu et al ., 1979M 12 40.5 50 600 48 15.5 4.17 NR Vers ch ae ve t al., 1978M 20 20-49 10J0 1950 48 7.1 1.85 I) .5 6 Vers ch ae ve t al. , 1979 'jM + F 119 0-70 I(X)0 ~ 107792 50 NR 0.611 NR Tonomura et al. 1983M 280 18-46 NR 31773 48 11.8 11.13 NR Brown et al., 1983M + F 20 NR 49 976 54 2.5 11.1 11.2 Ding et al ., 1983M 11 NR 200 2200 48 NR NR I).09 Clare et al., 1984M + F 3(14 NR 100 182911 48-51 NR 0.11 0.16 f Ga llo wa y t al., 1986M + F 14 31.6 100 1400 48 39.1 1.85 NR Monsalve nd Chlappe, 1987M + F 493 1-84 21~1 108950 48-56 6.6 ~ 0.15 b I).113 Benderet a[., 1989M 29 NR 86 2481 50 NR 0 NR Yardley-Jones et al., 1990M + F 10 21-40 200 2008 48 NR 0.2 NR Barquinero et al. 1993M + F 8 0-77 120 998 48 13.0 0.33 NR Richard et al. 1993

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


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160 D . A . E a s t m o n d e t a l. / M u t a t i o n R e s e a r c h 3 4 8 ( 1 9 95 ) 1 5 3 - 1 6 2

a n d 4 h y b r i d i z a t i o n s i g n a l s wa s 0 . 3 4 % ( s t a n d a r dd e v i a t i o n 0 . 2 4 %; r a n g e 0 - 1 . 1 % ) . S i n c e t h is s tu d yused on ly a s ing le c l a s s ica l sa te l l i t e p robe , hype r-d i p l o i d n u c l e i c a n n o t b e d i s t i n g u i s h e d f r o m s i mi -l a r a p p e a r i n g n u c l e i r e s u l t i n g f r o m c h r o m o s o m e

b r c a k a g e . H o w e v e r b a s e d o n t h e p r o p o r t io n s o fb r e a k s v s . n u me r i c a l c h a n g e s s e e n i n t h e c o n t r o l so f t h e p e s t i c i d e s t ud y, t h e p r o p o r t i o n o f t h e s en u c l e i r e p r e s e n t i n g h y p e r d i p l o i d y c a n b e e s t i-ma t e d a s ~ 4 4 % . Th i s r e s u l ts i n a n e s t i ma t e dh y p e r d i p l o i d f r e q u e n c y o f 0 . 1 5 % wh i c h i s s i m i la rt o t h a t s e e n i n t h e p e s t i c i d e s t u d y. Ba s e d o nt h e s e t wo FI S H s t u d i e s , t h e a v e r a g e f r e q u e n c y o fn u c l e i c o n t a i n i n g 3 o r 4 c o p i e s o f c h r o mo s o m e 1i n 4 8 h r c u l t u r e s wo u l d t h e n b e a p p r o x i ma t e l y0 . 1 6 % .

I f o n e a s s u m e s t h a t o n ly o n e c h r o m o s o m e a t at ime i s inw) lved in hype rd ip lo idy and tha t a l lc h r o mo s o m e s a r e e q u a l l y l ik e ly to b e i n v o lv e d inh y p e r d i p l o i d y, t h e n t h e f r e q u e n c y o f n u c le i c o n -t a i n i n g 3 o r 4 c o p i e s o f a s p e c i fi c c h r o mo s o m e f o rt h e s t u d i e s l i s t e d i n Ta b l e 3 c a n b e e s t i ma t e d a s0 . 2 0 % { 0 .1 7 % ( p o l y p l o i d y f r e q u e n c y ) + 0 . 0 3 2 %[ t h e to t a l h y p e r d i p l o i d f r e q u e n c y ( 0 . 7 3 %) d i v i d e db y t he 2 3 c h r o m o s o m e c o m p l e m e n t s i n hu m a ns ]} .I f t h e h i g h p o l y p l o i d f r e q u e n c y r e p o r t e d b y Ve r -schaev c c t a l. (1979) is om i t t ed , the ex pec tedf r e q u e n c y b e c o m e s 0 .1 3 1 % . G i v e n t h e n a t u r e o fthe e s t im a tes - va r i ab i l i ty in the d i f f e re n t expe r i -me n t s , d i f f e r e n t s t u d y p o p u l a t i o n s , q u e s t i o n a b l ea s s u mp t i o n s , l i m i t e d n u m b e r o f s tu d i e s , e tc . , t h ee s t i ma t e s p r o v i d e d f r o m t h e F I SH a n a l y s e s a n dc o n v e n t i o n a l m e t a p h a s e s a r c r e m a r k a b l y s i m i l a r.I n a g r e e me n t w i t h t h i s , d i r e c t c o mp a r i s o n s o fh y p e r d i p l o i d ( a n d b r e a k a g e ) f r e q u e n c i e s f o r s p e -c if ic c h r o m o s o m e s in o u r l a b o r a t o r y a n d o t h e r s(Podd ighe c t a l . , 1991 ; Hasegawa e t a l . , 1995)h a v e g e n e r a l l y y i e l d e d s i mi l a r f r e q u e n c i e s wh e nt h e r e s u l t s o f me t a p h a s e a n d i n t e r p h a s e c e l l s

h a ve b e e n c o m p a r e d . W h e n d i f f e r e n c e s b e t w e e ni n t e r p h a s e a n d m e t a p h a s e F I S H s t u d i e s h a v eb e e n s e e n i n o u r l a b o r a t o r y, t h e y t y p i c al l y h a v eb e e n t h o s e i n wh i c h h i g h e r n u m b e r s o f n u c l e iw i t h 3 a n d 4 c h r o m o s o m e c o p i e s we r e s e e n i n t h eme t a p h a s e p r e p a r a t i o n s , p o s s i b ly re f l e c t i n g t h econse rva t i sm in ou r sco r ing o r d i f f i cu l t i e s in d i s -t i n g u i s h i n g 2 a d j a c e n t d i p l o i d me t a p h a s e s f r o m ap o l y p l o i d mc t a p h a s c . O n e p o i n t t h a t s h o u l d b e

e mp h a s i z e d f r o m t h e a b o v e c a l c u l a t i o n s i s t h a tnuc le i exh ib i t ing 3 o r 4 hybr id iza t ion reg ions inFI SH s t u d i e s o f u n t r e a t e d c e l l s a l mo s t c e r t a i n l yr e p r e s e n t p o l y p l o i d c el l s r a t h e r t h a n n u c l e i h y -p e r d i p l o i d f o r a n i n d iv i d u a l c h r o m o s o m e . O t h e r -

wi s e , t h e o b s e r v e d h y p e r d i p l o i d f r e q u e n c i e s o f0 . 1 6 % f o r o n e c h r o m o s o m e m u l t i p li e d b y th e 2 3c h r o mo s o me p a i r s wo u l d r e s u l t i n a o v e r a l l h y -p c r d i p to i d f r e q u e n c y o f 3 . 7 % , a f r e q u e n c y v e r yd i f f e r e n t f r o m t h o s e s e e n i n mc t a p h a s e s t u d i e s .Th e r e s u l ts o f t h e s e a n a l y s e s i n d i c a t e t h a t i n s p it eo f v a r io u s c e l l u l a r a n d h y b r i d i z a t i o n a r t i f a c ts .F I SH c a n b e u s e d t o p r o v i d e r e a s o n a b l e e s t i -m a t e s o f t h e f r e q u e n c y o f n u m e r i c a l a l t e ra t i o n sin human ce l l s when cau t ion i s exe rc i sed in thesco r ing p rocess .

In summary, wc b e l i e v e t h a t i n t e r p h a s e a n a l y -s i s u s i n g F I S H w i t h c h r o m o s o m e - s p e c i f i c D N Ap r o b e s i s a v a l u a b l e c o mp l e me n t t o c o n v e n t i o n a lme t a p h a s e a n a l y s i s f o r d e t e c t i n g a n e u p l o i d y i nt h e s o m a t i c c e ll s o f h u ma n s a n d a n i ma l s . I n th i sa r t i c l e wc h a v e t r i e d t o d e s c r i b e ma n y o f t h ep o s s ib l e c e l l u l a r a n d h y b r i d i z a t i o n c o n d i t i o n s t h a tc a n a f f e c t t h e i n t e r p r e t a t i o n o f F I SH a n a l y s e s f o ra n e u p l o i d y. I n s p i te o f t h e s e p o t e n t i a l p r o b l e m s ,a n u m b e r o f s t u di e s h a ve d e m o n s t r a t e d t h a t F I S Hc a n b e a v a l u a b l e t o o l f o r i n v i t r o s c r e e n i n g o fa g e n t s w i t h a n e u p l o i d y - i n d u c i n g p o t e n t i a l a n d f o rd e t e c t i n g a n e u p l o i d y i n c h e mi c a l l y e x p o s e d h u -ma n p o p u l a t i o n s . W i t h e f f i c i e n t h y b r i d i z a t i o n sa n d c a u t i o u s s c o r i n g , i n t c r p h a s e s t u d i e s u s i n gF I S H t o d e t e c t a n e u p l o i d y (a n d b r e a k a g e ) c a np r o v i d e e s t i ma t e s o f c h r o m o s o m a l a l t e r a t i o n swh i c h c l o s el y r e f l e c t t h o s e s e e n d u r i n g m e t a p h a s ca n a l y s i s u s i n g e i t h e r F I SH o r c o n v e n t i o n a l a p -p r o a c h e s . Du e t o t h e s p e e d o f a n al y s is a n d t h ea b i li t y t o e v a l u a t e d i f f e r e n t c e l l ty p e s , c y t o g e n c t i ca n a l y s i s u s i n g F I SH s h o u l d c o n t r i b u t e s i g n i f i -c a n t l y t o o u r u n d e r s t a n d i n g o f th e e t i o l o g y a n d

c o n s e q u e n c e s o f a n e u p l o i d y a f f e c t i n g h u m a np o p u l a t i o n s .

Acknowledgments

We wo u l d l i k e t o t h a n k Sh e i l a Ga l l o wa y f o rh e r h e l p f u l d i s c u s s i o n s a n d f o r p r o v i d i n g u s w i t ht h e 4 8 h h y p e r d i p l o i d y a n d p o l y p l o i d y d a t a f r o m


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her popula t ion s tudies . F inanc ia l suppor t f romt he U. S . Env i r onm en t a l P r o t ec t i on Agency ( R820994-01-1) is grateful ly acknowledged.

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Communicated by S.M. Galloway

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