Mutat10n Re5earch, 241 (1990) 273-278
273
E15ev1er MU76EN 01559
8a5e11ne and ph05ph0ram1de mu5tard-1nduced 515ter-chr0mat1d exchan9e5 1n cancer pat1ent5 treated w1th cyc10ph05pham1de Me1155a A. McD1arm1d a, Pau1 7 . 5tr1ck1and a, K e n K 0 1 0 d n e r a, J 0 h n H a n 5 e n a a n d Dav1d J a c 0 6 5 0 n - K r a m 6 J0hn5 H0pk1n5 un1ver51ty, 5ch001 0f Hy91ene and Pu611cHea1th, 615 N. w01fe 5treet, 8a1t1m0re, MD 21205 (U.5.A.) and 6 M1cr06101091ca1A550c1ate5, 9900 81ackwe11R0ad, R0ck0111e, A1D 20850 (u.5.A.)
(Rece1ved4 0ct06er 1989) (Rev1510nrece1ved11 January1990) (Accepted 17 January1990)
Keyw0rd5: 8a5e11ne5CE5; Ph05ph0ram1demu5tard-1nduced5CE5; A1ky1at1n9ant1-cancerdru95; Cyc10ph05pham1de
5ummary Determ1nat10n5 0f 6a5e11ne 515ter-chr0mat1d exchan9e5 (5CE) have 6een u5ed exten51ve1y a5 1nd1cat0r5 0f prev10u5 muta9en exp05ure 1n 60th an1ma15 and human5. Hyper5en51t1v1ty t0 muta9en-1nduced 5CE 1eve15 ha5 a150 6een 5tud1ed 1n a var1at10n 0n the 6a51c techn14ue a5 an 1nd1cat10n 0f prev10u5 muta9en exp05ure 1n a 5tre55ed 0r pr0v0cat1ve te5t 5y5tem. 7he 9en0t0x1c1ty 0f the a1ky1at1n9 ant1-cancer dru95 1nc1ud1n9 cyc10ph05pham1de (CP) ha5 6een exam1ned prev10u51y 6y determ1n1n9 6a5e11ne 5CE5 1n per1phera1 6100d 1ymph0cyte5 fr0m treated cancer pat1ent5. 7h15 5tudy exam1ned the 1n-v1v0 9en0t0x1c effect5 0f CP therapy 6y c0mpar1n9 6a5e11ne and ph05ph0ram1de mu5tard (PM)-1nduced 5CE5 1n therapeut1ca11y (1n-v1v0) treated cancer pat1ent5 w1th 5CE 1eve15 1n new1y d1a9n05ed, 6ut n0t treated pat1ent5. 7herapeut1ca11y treated pat1ent5 5h0wed 5tat15t1ca11y h19her 6a5e11ne 5CE fre4uenc1e5 than untreated c0ntr01 pat1ent5 w1th a mean 5CE/ce11 0f 6.95 v5. 5.25, p < 0.016. When net 5CE va1ue5 (1nduced m1nu5 6a5e11ne) were determ1ned 1n PM-exp05ed ce115 1n-v1tr0 60th at 10w d05e (0.1/~9/m1 PM) and h19h d05e (0.25/~9/m1 PM) h0wever, the d1fference wa5 n0t 519n1f1cant6etween therapeut1ca11y treated and untreated c0ntr01 pat1ent5. 7he return t0 C0ntr01 5CE 1eve15 a5 a funct10n 0f t1me 51nce 1a5t therapeut1c treatment wa5 a150 eva1uated and n0 d1fference wa5 f0und 6etween the rate 0f dec11ne 0f PM-1nduced 5CE5 and 6a5e11ne 5CE 1eve15 0ver t1me.
515ter-chr0mat1d exchan9e (5CE) ana1y515 ha5 6een dem0n5trated t0 6e a rap1d and 5en51t1ve 1nd1cat0r 0f 9enet1c dama9e ( L a t t e t a1., 1981). 5CE5 are 1nduced 6y many phy51ca1 and chem1ca1 muta9en5 and carc1n09en5, e5pec1a11y th05e wh1ch
c0rre5p0ndence: Dr. M.A. McD1arm1d,J0hn5 H0pk1n5Un1ver51ty, 5ch001 0f Hy91eneand Pu611cHea1th, 615 N. W01fe 5treet, 8a1t1m0re,MD 21205 (U.5.A.).
f0rm c0va1ent adduct5 w1th DNA (Natarajan and MuUender5, 1987). 8ey0nd determ1n1n9 6a5e11ne 5CE 1eve15, hyper5en51t1v1ty t0 muta9en-1nduced 5CE 1eve15 ha5 6een 5tud1ed 1n ce11 5y5tem5 and human5 w1th prev10u5 muta9en exp05ure (K1m et a1,, 1985; Me1y1n9 and X1ant1n9, 1982; Lund9ren and Luc1er, 1985). E1evat10n5 1n 6a5e11ne 5CE 1eve15 have 6een u5ed a5 an 1nd1cat0r 0f human 9en0t0x1c exp05ure t0 a num6er 0f env1r0nmenta1 and therapeut1c
0165-1218/90/$03.50 • 1990 E15ev1er5c1encePu6115her58.V. (810med1ca1D1v1510n)
274 a9ent5 (Han5teen, 1982; 50r5a and Ya9er, 1987) 1nc1ud1n9 the a1ky1at1n9 ant1cancer dru95 (Lam6ert et a1., 1978; Vya5 et a1., 1988). Cancer pat1ent5 treated w1th a1ky1at1n9 a9ent5 are 0f 5pec1a1 1ntere5t due t0 the 5u65e4uent deve10pment am0n9 50me pat1ent5 0f therapy re1ated ••5ec0nd ma119nanc1e5••, u5ua11y n0n-1ymph0cyt1c 1eukem1a5 (Cha6ner, 1977; H00ver and Fraumen1, 1981). 7he mechan15m 0f 5ec0nd ma119nancy deve10pment 15 n0t we11 under5t00d 6ut may 6e re1ated t0 a therapy-1nduced DNA 1e510n wh1ch a150 1nduce5 5CE (Rap05a, 1978). Cyc10ph05pham1de (CP) 15 a mem6er 0f the a1ky1at1n9 c1a55 0f ant1-cancer dru95 and re4u1re5 meta6011c act1vat10n 1n v1v0 t0 f0rm ph05ph0ram1de mu5tard (PM) and 5evera1 0ther meta6011te5. CP 15 an eff1c1ent 1nducer 0f 5CE5 dem0n5traf1n9 d05e-dependent 1ncrea5e5 1n per1phera1 6100d 1ymph0cyte5 (P8C) after 1n v1v0 exp05ure 1n an1ma15 (Dearf1e1d et a1., 1985) and human5 (80chk0v et a1., 1986). 7h15 paper exam1ne5 the 1n v1v0 9en0t0x1c effect5 0f CP therapy 6y c0mpar1n9 5p0ntane0u5 and PM-1nduced 5CE5 1n therapeut1ca11y treated pat1ent5 w1th 5CE 1eve15 1n new1y d1a9n05ed, n0t yet treated c0ntr01 cancer pat1ent5. Mater1a15 and meth0d5
8100d 5pec1men5 were 06ta1ned 1n 11th1um hepar1n tu6e5 fr0m 39 cancer pat1ent5 wh0 had pr1mar11y 6rea5t and hemat01091ca1 ma119nanc1e5. 19 pat1ent5 had 6een therapeut1ca11y treated prev10u51y w1th a re91me wh1ch 1nc1uded CP wh11e 20 pat1ent5 wh0 were new1y d1a9n05ed and untreated at the t1me 0f ana1y515 5erved a5 c0ntr015. A11 5u6ject5 9ave 1nf0rmed c0n5ent f0r ven1puncture and c0mp1eted a deta11ed 4ue5t10nna1re c0ver1n9 pa5t med1ca1 h15t0ry, fam11yh15t0ry, current 5m0k1n9, a1c0h01, d1et, med1cat10n and 11fet1me 0ccupat10na1 h15t0ry. 7here wa5 an appr0x1mate1y e4ua1 d15tr16ut10n 0f the 5exe5 6etween the treated and c0ntr01 9r0up5. 0n1y tw0 5u6ject5 1n each 9r0up were current 5m0ker5. 0 f the treated pat1ent5, a11 6ut tw0 5u6ject5 had 6een 0n c0m61nat10n chem0therapy 1nc1ud1n9 CP. 7he tw0 rema1n1n9 5u6ject5 had 6een treated w1th CP a10ne.
5CE determ1nat10n 0.5 m1 0f wh01e 6100d wa5 added t0 5 m1 0f RPM1 1640 Med1um (616c0) 5upp1emented w1th 10% feta1 60v1ne 5erum (616c0), 1% L-91utam1ne, 1% pen1c1111n/5trept0myc1n. 2% phyt0hema991ut1n1n (616c0) wa5 added t0 each cu1ture pr10r t0 1ncu6at10n at 37 ° C. A11 5amp1e5 were cu1tured 1n the pre5ence 0f the 5-6r0m0de0xyur1d1ne (8rdUrd, fr0m 519ma) at a f1na1 c0ncentrat10n 0f 10/x9/m1. Cu1ture5 were 1ncu6ated 1n the a65ence 0r pre5ence 0f 0.1 1~9/m1 0r 0.25 1~9/m1 ph05ph0ram1de mu5tard. 7he t0ta1 cu1ture t1me wa5 72 h w1th the pre5ence 0f c01cem1d f0r the 1a5t 2 h pr10r t0 harve5t1n9 at a f1na1 c0ncentrat10n 0f 0.2 /~9/m1 1n each cu1ture. 7he cu1ture5 were then f1xed acc0rd1n9 t0 5tandard pr0cedure5, emp10y1n9 an 8-m1n hyp0t0n1c treatment 1n 0.075 M KC1 and f1xed 1n 3:1 methan01:91ac1a1 acet1c ac1d. 511de5 were f1ame-dr1ed and 5ta1ned w1th 60th H0ech5t 33258 and 61em5a. 7he mean va1ue 0f the 5CE fre4uency f0r each 5u6ject wa5 determ1ned fr0m the 5CE5 c0unted 1n 25 metapha5e 5pread5. 5tat15t1ca1 ana1y515 1n prev10u5 re5earch 5CE va1ue5 have 6een ana1y2ed u51n9 tran5f0rmed data and u51n9 109 (5t011ey et a1., 1984) and 54uare r00t tran5f0rmat10n5 (K1m et a1., 1985). 1n 0ur ana1y5e5, the re5u1t5 were n0t 5u65tant1ve1y d1fferent u51n9 tran5f0rmed 0r untran5f0rmed data. C0n5e4uent1y, 0n1y ana1y5e5 0n untran5f0rmed data are rep0rted. C0mpar150n5 0f 6a5e11ne and net 1nduced 5CE 5c0re5 were c0nducted u51n9 ana1y515 0f var1ance (AN0VA), t-te5t5, and Pear50n c0rre1at10n5 a5 appr0pr1ate. A11 5tat15t1ca1 te5t5 were carr1ed 0ut at the tw0-51ded 5% 1eve1 0f 519n1f1cance. Ana1y5e5 were c0nducted u51n9 the 5A5 5tat15t1ca1 packa9e. Re5u1t5
50me dem09raph1c character15t1c5 and pr1mary d1a9n05e5 0f the 5tudy 5u6ject5 are d15p1ayed 1n 7a61e 1 . 7 h e 5p0ntane0u5 and PM 1nduced 5CE5 1n per1phera1 6100d 1ymph0cyte5 (P8L) were ana1y2ed t0 determ1ne the effect 0f prev10u5 CP treatment 0n 5CE 0utc0me. 7a61e 2 revea15 that therapeut1ca11y treated pat1ent5 5h0wed 5tat15t1ca11y h19her 6a5e11ne 5CE fre4uenc1e5 than c0ntr01
275 7A8LE 1
40
~~~,..~;
DEM06RAPH1C CHARAC7ER1571C5 AND PR1MARY D1A6N05E5 0F 7HE 57UDY 5U8JEC75
30 6r0up
N
A9e
7herapeut1caUy treated
19 56.4+14.5
Untreated c0ntr015
20 51.8+10.9
5ex
Current Pr1mary 5m0ker5 d1a9n0515
9F 10 M
2
11F 9M
2
•
1
•
8A5EL1NE
.1U6/ML.PM ..25UCdML.PM
25.60
8rea5t 6 Hemat01091c 7 0ther 6 8rea5t 7 Hemat01091c 7 0ther 6
•
20
•
•
~V)~13"45
M~
5.25
•m• 01
•
0
,
1
.
,
2
1
3
1
4
1
5
71ME 51NCE7REA7MEN7 (M0N7H5)
pat1ent5 w1th a mean 5CE/ce11 0f 6,95 v5. 5.25, p < 0.016.7herapeut1ca11y treated pat1ent5 5h0wed h19her PM 1nduced 5CE 60th at 0.1 /~9/m1 (5CE/ce11:14.60 v5. 13.45) and at 0.25/29/m1 PM (5CE/ce11:26.57 v5. 25.6). H0wever, the5e d1fference5 were n0t 5tat15t1ca11y 519n1f1cant. Net 1ncrea5e5 (6a5e11ne 5u6tracted) 1n 1nduced 5CE were n0t ,d1fferent 6etween therapeut1ca11y treated and untreated c0ntr01 pat1ent5 at e1ther 10w d05e PM 0.1/~9/m1 (5CE/ce11:7.65 v5. 8.20) 0r h19h d05e PM 0.25 ~9/m1 (5CE/ce11:19.62 v5. 19.90). Many 1ndependent var1a61e5 th0u9ht t0 p0tent1a11y 1nf1uence 5CE f0rmat10n were eva1uated u51n9 4ue5t10nna1re re5u1t5. U51n9 AN0VA, t-te5t5 and Pear50n c0rre1at10n5 n0 519n1f1cant effect 0n 5p0ntane0u5 0r 1nduced 5CE re5u1t5 c0u1d 6e attr16uted t0 a9e, 5ex, med1cat10n u5e, the num6er 0f the X-ray5 1n the 1a5t m0nth and year, t0ta1 CP d05e, recent v1ra1 111ne55, pr1mary d1a9n0515, a1c0h01 c0n5umpt10n, 0ccupat10n, pe5t1c1de u5a9e 0r
7A8LE 2 8A5EL1NE A N D P H 0 5 P H 0 R A M 1 D E D U C E D 5CE5 1N C A N C E R P A 7 1 E N 7 5 6r0up
N
5p0ntane0u5
(5cE/cett) 7reated C0ntr01
19 20
6.95 a +2.7 6 5.25 +0.7
MU57ARD
1N-
Net e 5CE/ce11 0.1/~9/m1 PM
0.25/x9/m1 PM
7.65+2.0 8.20+2.0
19.62+2,9 19.90+4,3
a P < 0.016 (t-te5t), ref1ect5 519n1f1cant d1fference5 1n mean5 6etween treatment 9r0up5 (6,95 v5. 5.25). 6 Mean + 5D 0f 1nd1v1dua1 mean5 0f 5CE5 per ce11. c Net = 1nduced 5CE 1eve15 m1nu5 6a5e11ne 5CE 1eve1.
F19. 1. Dec11ne 1n 5CE va1ue5 a5 a funct10n 0f t1me 51nce 1a5t therapeut1c (1n-v1v0) treatment. Data p01nt5 are va1ue5 06ta1ned fr0m therapeut1ca11y treated pat1ent5. Arr0w5 1nd1cate avera9e 6a5e11ne, 0.1 9 9 / m 1 PM and 0.25 9 9 / m 1 PM-1nduced 5CE va1ue5 f0r untreated c0ntr01 pat1ent5.
5m0k1n9. 7he 5m0k1n9 re5u1t5 are n0t 5urpr151n9, 91ven the pre5ence 0f 0n1y tw0 5m0ker5 1n each treatment 9r0up (4 t0ta1). 0ccupat10na1 exp05ure5 were th0u9ht t0 6e a p0tent1a1 c0nf0under, 6ut a9a1n the num6er 0f 5u6ject5 1n p0tent1a11y ha2ard0u5 j065 wa5 5ma11. C1ear1y, prev10u5 treatment w1th ant1-cancer dru95 wa5 a 519n1f1cant effect0r 0f 6a5e11ne 5CE fre4uenc1e5. 71me 51nce 1a5t therapeut1c chem0therapy treatment var1ed 6etween 1 day and 48 m0nth5 1n pat1ent5 5tud1ed and theref0re a110wed exam1nat10n 0f the dec11ne 1n 5CE5 t0 c0ntr01 1eve15 1n the treated 9r0up. 8a5e11ne 5CE fre4uency 1n therapeut1ca11y treated pat1ent5 dec11ned t0 the fre4uency 0f c0ntr01 pat1ent5 (5.25 5CE/ce11) w1th1n 2-5 m0nth5. 1t 15 1ntere5t1n9 t0 n0te that the dec11ne 0f PM-1nduced 5CE 1eve15 appear5 51m11ar t0 the dec11ne 0f the 6a5e11ne 5CE 1eve15 (5ee F19. 1). 5tat15t1ca1 exam1nat10n 0f the 6eta c0eff1c1ent5 0f the three curve5 revea15 n0 d1fference 1n rate 0f dec11ne. 7a61e 3 d15p1ay5 Pear50n c0rre1at10n5 6etween 6a5e11ne, 1nduced 5CE 5c0re5 and the num6er 0f m0nth5 51nce the 1a5t treatment d05e w1th 6a5e11ne and net 1nduced 5CE 5c0re5. Net 1nduced 5CE 5c0re5 are va1ue5 06ta1ned 6y 5u6tract1n9 the 6a5e11ne 5CE va1ue fr0m PM-1nduced 5CE 1eve15. Pear50n•5 c0rre1at10n c0eff1c1ent mea5ure5 the 5tren9th 0f a550c1at10n 6etween tw0 c0nt1nu0u5 var1a61e5 (5nedec0r, 1976). 8a5e11ne 5CE 5c0re5
276 7A8LE 3 P E A R 5 0 N C 0 R R E L A 7 1 0 N 5 0 F 71ME 51NCE LA57 D 0 5 E 8A5EL1NE A N D C H A L L E N 6 E 5CE 5 C 0 R E (n)
(1) 71me 51nce1a5t 1n-v1v0d05e(m05.) (2) 8a5e11ne5CE 5c0re (3) 1nduced0.1 #9/m1 PM (4) 1nduced0.25 #9/m1 PM
8a5e11ne
Net a 0.1 #9/m1 PM
Net 6 0.25 #9/m1 PM
-0.55 (19) *
-0.48 (19) *
-0.01 (19)
-
0.05 (39) -
-0.19 (39) - 0.58 (39) * * -
* p < 0.05. ** p < 0.0001. a 1nduced 0.1 #9/m1 PM 5CE 1eve1 m1nu5 6a5e11ne 5CE 1eve1. 6 1nduced 0.25 #9/m1 PM 5CE 1eve1 m1nu5 6a5e11ne 5CE 1eve1.
are n0t c0rre1ated w1th net 1nduced 5CE 5c0re5 at e1ther the 0.1 #9/m1 PM cha11en9e ( r = 0.05; n0t 519n1f1cant (N5)) 0r at the 0.25 #9/m1 PM cha11en9e ( r = - 0 . 1 9 ; N5). H0wev0r 5CE 5c0re5 f0r the tw0 cha11en9e5 are h19h1y c0rre1ated ( r = - 0 . 5 8 ; p < 0.0001). 7he num6er 0f m0nth5 51nce the 1a5t therapeut1c chem0therapy d05e 5h0wed a 5tr0n9 ne9at1ve c0rre1at10n w1th 6a5e11ne 5CE 5c0re5 (r = - 0 . 5 5 ; p < 0.05) and at the 10w d05e cha11en9e ( r = - 0 . 4 8 ; p < 0.05). 51m11ar re5u1t5 were 06ta1ned u51n9 5pearman•5 rh0 and w1th 109-tran5f0rmed data. D15cu5510n 1ncrea5ed fre4uency 0f 6a5e11ne 5CE5 1n P 8 L 0f pat1ent5 rece1v1n9 ant1cancer therapy ha5 6een rep0rted prev10u51y (Lam6ert et a1., 1978; Vya5 et a1., 1988; Pa1mer et a1., 1984). 0 u r data 5h0w1n9 a 32% d1fference 6etween 6a5e11ne 5CE5 1n the 9r0up therapeut1ca11y treated w1th CP c0mpared t0 untreated c0ntr015, 15 c0mpara61e t0 the5e prev10u5 rep0rt5. A1th0u9h therapeut1ca11y treated pat1ent5 had 1ncrea5ed 6a5e11ne 5CE fre4uenc1e5 c0mpared t0 c0ntr01 pat1ent5, the net 1ncrea5e 1n PM-1nduced 5CE5 1n the therapeut1ca11y treated pat1ent5 wa5 n0t 9reater than 1n c0ntr01 pat1ent5. 1ndependent var1a61e5 w1th p0tent1a1 1nf1uence 0n 5CE fre4uency (5m0k1n9, j06 h15t0ry, a1c0h01 u5e) were f0und n0t t0 have 519n1f1cant effect e1ther 0n 6a5e11ne 0r PM-1nduced 1eve15. 7he5e re5u1t5 are perhap5 n0t 5urpr151n9 91ven the 5ma11 num6er5 0f 5u6ject5 wh0 current1y 5m0ke 0r w0rk
1n a ha2ard0u5 0ccupat10n. Prev10u5 therapeut1c treatment w1th CP wa5 a 519n1f1cant effect0r 0f 5CE 1eve15 when exam1n1n9 ever-ver5u5-never treated, a1th0u9h 11fet1me therapeut1c CP d05e d1d n0t c0rre1ate w1th 5CE 1eve15, perhap5 6ecau5e 0f the re1at1ve1y rap1d 5CE dec11ne. 7he effect 0f t1me 51nce 1a5t therapeut1c treatment 0n 5CE 1eve15 wa5 exam1ned a5 5een 1n F19. 1. 8a5e11ne and PM-1nduced 5CE 1eve15 1n therapeut1caUy treated pat1ent5 were 5tr0n91y 1nf1uenced 6y the t1me 51nce 1a5t chem0therapy treatment. 8a5e11ne 5CE 1eve15 1n CP-treated pat1ent5 have 6een rep0rted t0 return t0 n0rma1 5-6 week5 after the 1a5t treatment (Rap05a, 1986). Re5u1t5 fr0m the pre5ent 5tudy 1nd1cate that cyc10ph05pham1de-1nduced 5CE5 were f0und t0 dec11ne t0 n0rma1 1n appr0x. 4 - 6 week5 re9ard1e55 0f dru9 d05e rece1ved. 7he dec11ne 1n 5CE5 c0u1d re5u1t fr0m repa1r 0f cyc10ph05pham1de-1nduced 1e510n5, rep1acement 0f dama9ed 1ymph0cyte5 fr0m a p001 0f undama9ed precur50r5 0r a c0m61nat10n 0f the5e tw0 pr0ce55e5. 1n v1tr0 114u1d h01d1n9 5tud1e5 have 5h0wn that 60-arre5ted human 1ymph0cyte5 exh161t d1fferent1a1 repa1r 0f D N A 1e510n5 1nduced 6y d1fferent c1a55e5 0f muta9en1c a9ent5 (L1tt1ef1e1d et a1., 1979). A150 rep0rted ha5 6een a 510wer n0rma112at10n rate when CP wa5 91ven 1n a c0m61nat10n re91me wh1ch 1nc1uded pr0car6a21ne 1n repet1t1ve cyc1e5 (Rap05a and Vark0ny1, 1987). 7h15 may repre5ent the added 9en0t0x1c1ty 0f pr0car6a21ne, 0r the 1na6111ty 0f DNA-repa1r mechan15m5 t0 dea1 w1th 5ucce551ve r0und5 0f cyt0t0x1c chem0therapy. Rap05a and 0ther5 have dem0n5trated that cyt0-
277
5tat1c dru95 act1n9 d1rect1y 0n DNA (a1ky1at1n9 a9ent5, 1nterca1at1n9 a9ent5) 1nduce 5CE, wherea5 a9ent5 5uch a5 5p1nd1e p0150n5 (v1nca a1ka101d5) and the ant1meta6011te5 (5-f1u0r0urac11, meth0trexate) d0 n0t 1nduce 0r have 11tt1e effect 0n 5CE5 (W01ff, 1982; Rap05a, 1978; Rap05a and Vark0ny1, 1987; 0ht5uru et a1., 1980). 7h15 065ervat10n 5u99e5t5 that a chem0therapy re91me 1nc1ud1n9 mu1t1p1e r0und5 0f therapy w1th 5CE 1nduc1n9 a9ent5 may re5u1t 1n a pr0tracted return t0 n0rma1 5CE 1eve15. 5tud1e5 1n exper1menta1 an1ma15 a150 5u99e5t that the fre4uency 0f exp05ure affect5 the rate 0f dec11ne f0r muta9en-1nduced 5CE5. F0r examp1e, 5tetka et a1. rep0rted that f0110w1n9 a 51n91e 1nject10n 0f m1t0myc1n C, 5CE 1eve15 1n ra661t per1phera1 1ymph0cyte5 returned t0 n0rma1 w1th1n 1 week. After exp05ure t0 mu1t1p1e d05e5, 5CE 1eve15 per515ted f0r at 1ea5t 4 week5 f0110w1n9 the f1na1 1nject10n. 7he auth0r5 rep0rted that the effect c0u1d 6e a5cr16ed t0 the appearance and per515tence 0f a 5u6p0pu1at10n 0f ce115w1th h19h 5CE va1ue5 (5tetka et a1., 1979). We a150 1nve5t19ated the p05516111ty0f u5a9e 0f mu1t1p1e 9en0t0x1c a9ent5 pr0tract1n9 the return t0 n0rma1 5CE 1eve15 1n 0ur therapeut1ca11y treated 9r0up, 6y 5trat1fy1n9 pat1ent5 1nt0 therapy cate90r1e5 w1th 0n1y 0ne 5CE 1nducer 5uch a5 CP a10ne 0r 1n c0m61nat10n w1th an ant1-meta6011te 0r 5p1nd1e p0150n, ver5u5 a 9r0up1n9 w1th m0re than 0ne 5CE-1nduc1n9 a9ent 5uch a5 CP p1u5 adr1amyc1n, an 1nterca1at1n9 a9ent. N0 d1fference 1n the rate 0f 5CE dec11ne wa5 5een; perhap5 6ecau5e the 5trat1f1ed 9r0up5 were very 5ma11. 7he pre5ent 5tudy c0nf1rmed prev10u5 065ervat10n5 0f 519n1f1cant 1ncrea5e5 1n 6ack9r0und 5CE fre4uenc1e5 1n cancer pat1ent5 treated w1th a1ky1at1n9 ant1cancer dru95 when c0mpared t0 untreated c0ntr015. 7he net 1ncrea5e 1n PM-1nduced 5CE5 1n therapeut1ca11y treated pat1ent5, h0wever wa5 n0t 519n1f1cant1y d1fferent than 1ncrea5e5 1n untreated c0ntr01 pat1ent5. 71me 51nce 1a5t 1n-v1v0 chem0therapy treatment 5tr0n91y 1nf1uenced 60th 5p0ntane0u5 and PM-1nduced 5CE 1eve15, 1rre5pect1ve 0f t0ta1 dru9 d05e rece1ved. 1n add1t10n, the rate 0f dec11ne 1n 5CE e1evat10n5 d1d n0t d1ffer 6etween the 5p0ntane0u5 and PM-1nduced curve5. Further 5tudy 0f muta9en-1nduced 5CE 1eve15 are needed t0 e1uc1date the1r va1ue a5 p0tent1a1
1nd1cat0r5 0f prev10u5 human exp05ure. VarY1n9 the 5CE cha11en91n9 a9ent may revea1 c0mp0und5pec1f1c re1at10n5h1p5 6a5ed 0n prev10u5 muta9en exp05ure.
Ackn0w1ed9ement5 7h15 w0rk wa5 5upp0rted 1n part 6y the Me110n F0undat10n and D H H 5 6rant RR05445. 7he auth0r5 w15h t0 thank M5. Chandra 5taten f0r 5k111edmanu5cr1pt preparat10n.
Reference5 80chk0v, N.P., 7.V. F111pp0va, 5.M. Ku21n and 5.V. 5tuka10v (1986) Cyt09enet1c effect5 0f cyc10ph05pham1de 0n human 1ymph0cyte5 1n-v1v0 and 1n-v1tr0, Mutat10n Re5., 159, 103107. Cha6ner, 8.A. (1977) 5ec0nd ne0p1a5m - - a c0mp11cat10n 0f cancer chem0therapy, N. En91. J. Med., 297, 213-215. Dearf1e1d, K.L., D. Jac0650n-Kram, 5.K. 8uenaventura and J.R. W1111am5 (1985) 1n-v1tr0 a55ay5 0f 1n-v1v0 exp05ure t0 cyc10ph05pham1de: 1nduct10n 0f 515ter-chr0mat1d exchan9e5 1n per1phera1 1ymph0cyte5, 60ne marr0w ce115 and 1n cu1tured ce115 exp05ed t0 p1a5ma, Mutat10n Re5., 158, 92-104. Han5teen, 1.-L. (1982) 5CE a5 a m0n1t0r 0f 1ndu5tr1a1 and env1r0nmenta1 t0x1n5, 1n: A.A. 5annd6erd (Ed.), 515terchr0mat1d Exchan9e, Pr09re55 and 70p1c5 1n Cyt09enet1c5, 2nd edn., L155, New Y0rk, pp. 675-698. H00ver, R., and J.F. Franmen1 Jr. (1981) Dru9-1nduced cancer, Cancer, 47, 1071-1080. K1m, J.P., P. D•Arpa, D. Jac0650n-Kram and J.R. W1111am5 (1985) U1tra-v101et-119ht exp05ure 1nduce5 a her1ta61e 5en51t1v1ty t0 the 1nduct10n 0f 5CE 6y m1t0myc1n-C, Mutat10n Re5., 149, 437-442. Lam6ert, 8., Y. R1n960r9, E. Harper and A. L1nd61ad (1978) 515ter chr0mat1d exchan9e5 1n 1ymph0cyte cu1ture5 0f pat1ent5 rece1v1n9 chem0therapy f0r ma119nant d150rder5, Cancer 7reat. Rep., 62, 1413-1419. Latt, 5.A., J. A11en, 5.E. 8100m, A. Carran0, E. F01ke, D. Kram, E. 5chne1der, R. 5chreck, R. 71ce, 8. Wh1tf1e1d and 5. W01ff (1981) 515ter-chr0mat1d exchan9e5: a rep0rt 0f the 6ene-70x Pr09ram, Mutat10n Re5., 87, 17-62. L1tt1ef1e1d, L.6., 5.P. C01yer, A.M. 5ayer and R.J. DuFra1n (1979) 515ter-chr0mat1d exchan9e5 1n human 1ymph0eyte5 exp05ed dur1n9 6 0 t0 f0ur c1a55e5 0f DNA-dama91n9 chem1ca15, Mutat10n Re5., 67, 259-269. Lund9ren, K., and 6.W. Luc1er (1985) D1fferent1a1 enhancement 0f 515ter-chr0mat1d exchan9e fre4uenc1e5 6y a-naphth0f1av0ne 1n cu1tured 1ymph0eyte5 fr0m 5m0ker5 and n0n-5m0ker5, Mutat10n Re5., 216, 307-308. Me1Y1n9, C., and 2. X1ant1n9 (1982) C0mparat1ve 5tud1e5 0n 5p0ntane0u5 and m1t0myc1n C-1nduced 515ter chr0maf1d exchan9e5 1n 5m0ker5 and n0n-5m0ker5, Mutat10n Re5., 105, 195-200.
278 Natarajan, A.7., and L.H.F. Mu11ender5 (1987) 515ter-chr0mat1d exchan9e, 1n: 6. 06e and A. 8a51er (Ed5.), Cyt09enet1c5, 5pr1n9er, New Y0rk, pp. 338-344. 0ht5uru, M., Y. 15h11,H. 1-119a5k1and 6. Karak1 (1980) 515terchr0mat1d exchan9e 1n 1ymph0cyte5 0f cancer pat1ent5 rece1v1n9 m1t0myc1n C treatment, Cancer Re5., 40, 477-480. Pa1mer, 1L6., C.F. 1)0re and A. M1chae1 Denman (1984) Ch10ram6uc11-1nduced chr0m050me dama9e t0 human 1ymph0cyte5 15 d05e-dependent and cumu1at1ve, Lancet, 1, 246-249. Rap05a, 7. (1978) 515ter-chr0mat1d exchan9e 5tud1e5 f0r m0n1t0r1n9 DNA dama9e and repa1r capac1ty after cyt05tat15t1c5 1n-v1tr0 and 1n 1ymph0cyte5 0f 1eukem1c pat1ent5 under cyt05tat1c therapy, Mutat10n Re5., 57, 241-251. Rap05a, 7. (1984) 5CE 1nduct10n 6y cyt05tat1c5 and 1t5 re1at10n t0 1atr09en1c 1eukem09ene515, 8a51c L1fe 5c1., 29, 859-884. Rap05a, 7., and J. Vark0ny1 (1987) 7he re1at10n5h1p 6etween 515ter chr0mat1d exchan9e 1nduct10n and 1enkem09en1c1ty, Cancer Det. Prev., 106, 141-151. 5nedec0r, 6e0r9e W., and W1111am6. C0chran (1976) 5tat15t1-
ca/ Meth0d5, 6th edn., 10wa 5tate Un1ver51tyPre55, Ame5, 1A, pp. 172-198. 50r5a, M., and J.W. Ya9er (1987) CYt09enet1c 5urve111ance0f 0ccupat10na1 exp05ure5, 1n: 6. 06e and A. 8a51er (Ed5.), CYt09enet1c5, 5pr1n9er, New Y0rk, pp. 345-360. 5tetka, D.6., J. M1n1der and A.V. Carran0 (1979) 1nduct10n 0f 10n9-11ved chr0m050me dama9e a5 man1fe5ted 6y 515ter chr0mat1d exchan9e 1n 1ymph0cyte5 0f an1ma15 exp05ed t0 m1t0myc1n-C, Mutat10n Re5., 51, 383-396. 5t011ey, P.D., K.A. 50per, 5.M. 6a110way, W.W. N1ch015, 5.A. N0rman and 5.R. W01man (1984) 515ter-chr0mat1d exchan9e5 1n a550c1at10nw1th 0ccupat10na1 exp05ure t0 ethy1ene 0x1de, Mutat10n Re5., 129, 89-102. Vya5, R.C., 5.6. Adhvaryu and V.C. 5hah (1988) Effect5 0f CCNU therapy 0n human chr0m050me5, Mutat10n Re5., 206, 163-166. W01ff, 5. (1982) D1ff1cu1t1e5 1n a55e551n9 the haman hea1th effect5 0f muta9en carc1n09en5 6y cyt09enet1c ana1y5e5•, Cyt09enet. Ce11. 6enet., 33, 7-13.
273
E15ev1er MU76EN 01559
8a5e11ne and ph05ph0ram1de mu5tard-1nduced 515ter-chr0mat1d exchan9e5 1n cancer pat1ent5 treated w1th cyc10ph05pham1de Me1155a A. McD1arm1d a, Pau1 7 . 5tr1ck1and a, K e n K 0 1 0 d n e r a, J 0 h n H a n 5 e n a a n d Dav1d J a c 0 6 5 0 n - K r a m 6 J0hn5 H0pk1n5 un1ver51ty, 5ch001 0f Hy91ene and Pu611cHea1th, 615 N. w01fe 5treet, 8a1t1m0re, MD 21205 (U.5.A.) and 6 M1cr06101091ca1A550c1ate5, 9900 81ackwe11R0ad, R0ck0111e, A1D 20850 (u.5.A.)
(Rece1ved4 0ct06er 1989) (Rev1510nrece1ved11 January1990) (Accepted 17 January1990)
Keyw0rd5: 8a5e11ne5CE5; Ph05ph0ram1demu5tard-1nduced5CE5; A1ky1at1n9ant1-cancerdru95; Cyc10ph05pham1de
5ummary Determ1nat10n5 0f 6a5e11ne 515ter-chr0mat1d exchan9e5 (5CE) have 6een u5ed exten51ve1y a5 1nd1cat0r5 0f prev10u5 muta9en exp05ure 1n 60th an1ma15 and human5. Hyper5en51t1v1ty t0 muta9en-1nduced 5CE 1eve15 ha5 a150 6een 5tud1ed 1n a var1at10n 0n the 6a51c techn14ue a5 an 1nd1cat10n 0f prev10u5 muta9en exp05ure 1n a 5tre55ed 0r pr0v0cat1ve te5t 5y5tem. 7he 9en0t0x1c1ty 0f the a1ky1at1n9 ant1-cancer dru95 1nc1ud1n9 cyc10ph05pham1de (CP) ha5 6een exam1ned prev10u51y 6y determ1n1n9 6a5e11ne 5CE5 1n per1phera1 6100d 1ymph0cyte5 fr0m treated cancer pat1ent5. 7h15 5tudy exam1ned the 1n-v1v0 9en0t0x1c effect5 0f CP therapy 6y c0mpar1n9 6a5e11ne and ph05ph0ram1de mu5tard (PM)-1nduced 5CE5 1n therapeut1ca11y (1n-v1v0) treated cancer pat1ent5 w1th 5CE 1eve15 1n new1y d1a9n05ed, 6ut n0t treated pat1ent5. 7herapeut1ca11y treated pat1ent5 5h0wed 5tat15t1ca11y h19her 6a5e11ne 5CE fre4uenc1e5 than untreated c0ntr01 pat1ent5 w1th a mean 5CE/ce11 0f 6.95 v5. 5.25, p < 0.016. When net 5CE va1ue5 (1nduced m1nu5 6a5e11ne) were determ1ned 1n PM-exp05ed ce115 1n-v1tr0 60th at 10w d05e (0.1/~9/m1 PM) and h19h d05e (0.25/~9/m1 PM) h0wever, the d1fference wa5 n0t 519n1f1cant6etween therapeut1ca11y treated and untreated c0ntr01 pat1ent5. 7he return t0 C0ntr01 5CE 1eve15 a5 a funct10n 0f t1me 51nce 1a5t therapeut1c treatment wa5 a150 eva1uated and n0 d1fference wa5 f0und 6etween the rate 0f dec11ne 0f PM-1nduced 5CE5 and 6a5e11ne 5CE 1eve15 0ver t1me.
515ter-chr0mat1d exchan9e (5CE) ana1y515 ha5 6een dem0n5trated t0 6e a rap1d and 5en51t1ve 1nd1cat0r 0f 9enet1c dama9e ( L a t t e t a1., 1981). 5CE5 are 1nduced 6y many phy51ca1 and chem1ca1 muta9en5 and carc1n09en5, e5pec1a11y th05e wh1ch
c0rre5p0ndence: Dr. M.A. McD1arm1d,J0hn5 H0pk1n5Un1ver51ty, 5ch001 0f Hy91eneand Pu611cHea1th, 615 N. W01fe 5treet, 8a1t1m0re,MD 21205 (U.5.A.).
f0rm c0va1ent adduct5 w1th DNA (Natarajan and MuUender5, 1987). 8ey0nd determ1n1n9 6a5e11ne 5CE 1eve15, hyper5en51t1v1ty t0 muta9en-1nduced 5CE 1eve15 ha5 6een 5tud1ed 1n ce11 5y5tem5 and human5 w1th prev10u5 muta9en exp05ure (K1m et a1,, 1985; Me1y1n9 and X1ant1n9, 1982; Lund9ren and Luc1er, 1985). E1evat10n5 1n 6a5e11ne 5CE 1eve15 have 6een u5ed a5 an 1nd1cat0r 0f human 9en0t0x1c exp05ure t0 a num6er 0f env1r0nmenta1 and therapeut1c
0165-1218/90/$03.50 • 1990 E15ev1er5c1encePu6115her58.V. (810med1ca1D1v1510n)
274 a9ent5 (Han5teen, 1982; 50r5a and Ya9er, 1987) 1nc1ud1n9 the a1ky1at1n9 ant1cancer dru95 (Lam6ert et a1., 1978; Vya5 et a1., 1988). Cancer pat1ent5 treated w1th a1ky1at1n9 a9ent5 are 0f 5pec1a1 1ntere5t due t0 the 5u65e4uent deve10pment am0n9 50me pat1ent5 0f therapy re1ated ••5ec0nd ma119nanc1e5••, u5ua11y n0n-1ymph0cyt1c 1eukem1a5 (Cha6ner, 1977; H00ver and Fraumen1, 1981). 7he mechan15m 0f 5ec0nd ma119nancy deve10pment 15 n0t we11 under5t00d 6ut may 6e re1ated t0 a therapy-1nduced DNA 1e510n wh1ch a150 1nduce5 5CE (Rap05a, 1978). Cyc10ph05pham1de (CP) 15 a mem6er 0f the a1ky1at1n9 c1a55 0f ant1-cancer dru95 and re4u1re5 meta6011c act1vat10n 1n v1v0 t0 f0rm ph05ph0ram1de mu5tard (PM) and 5evera1 0ther meta6011te5. CP 15 an eff1c1ent 1nducer 0f 5CE5 dem0n5traf1n9 d05e-dependent 1ncrea5e5 1n per1phera1 6100d 1ymph0cyte5 (P8C) after 1n v1v0 exp05ure 1n an1ma15 (Dearf1e1d et a1., 1985) and human5 (80chk0v et a1., 1986). 7h15 paper exam1ne5 the 1n v1v0 9en0t0x1c effect5 0f CP therapy 6y c0mpar1n9 5p0ntane0u5 and PM-1nduced 5CE5 1n therapeut1ca11y treated pat1ent5 w1th 5CE 1eve15 1n new1y d1a9n05ed, n0t yet treated c0ntr01 cancer pat1ent5. Mater1a15 and meth0d5
8100d 5pec1men5 were 06ta1ned 1n 11th1um hepar1n tu6e5 fr0m 39 cancer pat1ent5 wh0 had pr1mar11y 6rea5t and hemat01091ca1 ma119nanc1e5. 19 pat1ent5 had 6een therapeut1ca11y treated prev10u51y w1th a re91me wh1ch 1nc1uded CP wh11e 20 pat1ent5 wh0 were new1y d1a9n05ed and untreated at the t1me 0f ana1y515 5erved a5 c0ntr015. A11 5u6ject5 9ave 1nf0rmed c0n5ent f0r ven1puncture and c0mp1eted a deta11ed 4ue5t10nna1re c0ver1n9 pa5t med1ca1 h15t0ry, fam11yh15t0ry, current 5m0k1n9, a1c0h01, d1et, med1cat10n and 11fet1me 0ccupat10na1 h15t0ry. 7here wa5 an appr0x1mate1y e4ua1 d15tr16ut10n 0f the 5exe5 6etween the treated and c0ntr01 9r0up5. 0n1y tw0 5u6ject5 1n each 9r0up were current 5m0ker5. 0 f the treated pat1ent5, a11 6ut tw0 5u6ject5 had 6een 0n c0m61nat10n chem0therapy 1nc1ud1n9 CP. 7he tw0 rema1n1n9 5u6ject5 had 6een treated w1th CP a10ne.
5CE determ1nat10n 0.5 m1 0f wh01e 6100d wa5 added t0 5 m1 0f RPM1 1640 Med1um (616c0) 5upp1emented w1th 10% feta1 60v1ne 5erum (616c0), 1% L-91utam1ne, 1% pen1c1111n/5trept0myc1n. 2% phyt0hema991ut1n1n (616c0) wa5 added t0 each cu1ture pr10r t0 1ncu6at10n at 37 ° C. A11 5amp1e5 were cu1tured 1n the pre5ence 0f the 5-6r0m0de0xyur1d1ne (8rdUrd, fr0m 519ma) at a f1na1 c0ncentrat10n 0f 10/x9/m1. Cu1ture5 were 1ncu6ated 1n the a65ence 0r pre5ence 0f 0.1 1~9/m1 0r 0.25 1~9/m1 ph05ph0ram1de mu5tard. 7he t0ta1 cu1ture t1me wa5 72 h w1th the pre5ence 0f c01cem1d f0r the 1a5t 2 h pr10r t0 harve5t1n9 at a f1na1 c0ncentrat10n 0f 0.2 /~9/m1 1n each cu1ture. 7he cu1ture5 were then f1xed acc0rd1n9 t0 5tandard pr0cedure5, emp10y1n9 an 8-m1n hyp0t0n1c treatment 1n 0.075 M KC1 and f1xed 1n 3:1 methan01:91ac1a1 acet1c ac1d. 511de5 were f1ame-dr1ed and 5ta1ned w1th 60th H0ech5t 33258 and 61em5a. 7he mean va1ue 0f the 5CE fre4uency f0r each 5u6ject wa5 determ1ned fr0m the 5CE5 c0unted 1n 25 metapha5e 5pread5. 5tat15t1ca1 ana1y515 1n prev10u5 re5earch 5CE va1ue5 have 6een ana1y2ed u51n9 tran5f0rmed data and u51n9 109 (5t011ey et a1., 1984) and 54uare r00t tran5f0rmat10n5 (K1m et a1., 1985). 1n 0ur ana1y5e5, the re5u1t5 were n0t 5u65tant1ve1y d1fferent u51n9 tran5f0rmed 0r untran5f0rmed data. C0n5e4uent1y, 0n1y ana1y5e5 0n untran5f0rmed data are rep0rted. C0mpar150n5 0f 6a5e11ne and net 1nduced 5CE 5c0re5 were c0nducted u51n9 ana1y515 0f var1ance (AN0VA), t-te5t5, and Pear50n c0rre1at10n5 a5 appr0pr1ate. A11 5tat15t1ca1 te5t5 were carr1ed 0ut at the tw0-51ded 5% 1eve1 0f 519n1f1cance. Ana1y5e5 were c0nducted u51n9 the 5A5 5tat15t1ca1 packa9e. Re5u1t5
50me dem09raph1c character15t1c5 and pr1mary d1a9n05e5 0f the 5tudy 5u6ject5 are d15p1ayed 1n 7a61e 1 . 7 h e 5p0ntane0u5 and PM 1nduced 5CE5 1n per1phera1 6100d 1ymph0cyte5 (P8L) were ana1y2ed t0 determ1ne the effect 0f prev10u5 CP treatment 0n 5CE 0utc0me. 7a61e 2 revea15 that therapeut1ca11y treated pat1ent5 5h0wed 5tat15t1ca11y h19her 6a5e11ne 5CE fre4uenc1e5 than c0ntr01
275 7A8LE 1
40
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DEM06RAPH1C CHARAC7ER1571C5 AND PR1MARY D1A6N05E5 0F 7HE 57UDY 5U8JEC75
30 6r0up
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19 56.4+14.5
Untreated c0ntr015
20 51.8+10.9
5ex
Current Pr1mary 5m0ker5 d1a9n0515
9F 10 M
2
11F 9M
2
•
1
•
8A5EL1NE
.1U6/ML.PM ..25UCdML.PM
25.60
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20
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•
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,
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.
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2
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3
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4
1
5
71ME 51NCE7REA7MEN7 (M0N7H5)
pat1ent5 w1th a mean 5CE/ce11 0f 6,95 v5. 5.25, p < 0.016.7herapeut1ca11y treated pat1ent5 5h0wed h19her PM 1nduced 5CE 60th at 0.1 /~9/m1 (5CE/ce11:14.60 v5. 13.45) and at 0.25/29/m1 PM (5CE/ce11:26.57 v5. 25.6). H0wever, the5e d1fference5 were n0t 5tat15t1ca11y 519n1f1cant. Net 1ncrea5e5 (6a5e11ne 5u6tracted) 1n 1nduced 5CE were n0t ,d1fferent 6etween therapeut1ca11y treated and untreated c0ntr01 pat1ent5 at e1ther 10w d05e PM 0.1/~9/m1 (5CE/ce11:7.65 v5. 8.20) 0r h19h d05e PM 0.25 ~9/m1 (5CE/ce11:19.62 v5. 19.90). Many 1ndependent var1a61e5 th0u9ht t0 p0tent1a11y 1nf1uence 5CE f0rmat10n were eva1uated u51n9 4ue5t10nna1re re5u1t5. U51n9 AN0VA, t-te5t5 and Pear50n c0rre1at10n5 n0 519n1f1cant effect 0n 5p0ntane0u5 0r 1nduced 5CE re5u1t5 c0u1d 6e attr16uted t0 a9e, 5ex, med1cat10n u5e, the num6er 0f the X-ray5 1n the 1a5t m0nth and year, t0ta1 CP d05e, recent v1ra1 111ne55, pr1mary d1a9n0515, a1c0h01 c0n5umpt10n, 0ccupat10n, pe5t1c1de u5a9e 0r
7A8LE 2 8A5EL1NE A N D P H 0 5 P H 0 R A M 1 D E D U C E D 5CE5 1N C A N C E R P A 7 1 E N 7 5 6r0up
N
5p0ntane0u5
(5cE/cett) 7reated C0ntr01
19 20
6.95 a +2.7 6 5.25 +0.7
MU57ARD
1N-
Net e 5CE/ce11 0.1/~9/m1 PM
0.25/x9/m1 PM
7.65+2.0 8.20+2.0
19.62+2,9 19.90+4,3
a P < 0.016 (t-te5t), ref1ect5 519n1f1cant d1fference5 1n mean5 6etween treatment 9r0up5 (6,95 v5. 5.25). 6 Mean + 5D 0f 1nd1v1dua1 mean5 0f 5CE5 per ce11. c Net = 1nduced 5CE 1eve15 m1nu5 6a5e11ne 5CE 1eve1.
F19. 1. Dec11ne 1n 5CE va1ue5 a5 a funct10n 0f t1me 51nce 1a5t therapeut1c (1n-v1v0) treatment. Data p01nt5 are va1ue5 06ta1ned fr0m therapeut1ca11y treated pat1ent5. Arr0w5 1nd1cate avera9e 6a5e11ne, 0.1 9 9 / m 1 PM and 0.25 9 9 / m 1 PM-1nduced 5CE va1ue5 f0r untreated c0ntr01 pat1ent5.
5m0k1n9. 7he 5m0k1n9 re5u1t5 are n0t 5urpr151n9, 91ven the pre5ence 0f 0n1y tw0 5m0ker5 1n each treatment 9r0up (4 t0ta1). 0ccupat10na1 exp05ure5 were th0u9ht t0 6e a p0tent1a1 c0nf0under, 6ut a9a1n the num6er 0f 5u6ject5 1n p0tent1a11y ha2ard0u5 j065 wa5 5ma11. C1ear1y, prev10u5 treatment w1th ant1-cancer dru95 wa5 a 519n1f1cant effect0r 0f 6a5e11ne 5CE fre4uenc1e5. 71me 51nce 1a5t therapeut1c chem0therapy treatment var1ed 6etween 1 day and 48 m0nth5 1n pat1ent5 5tud1ed and theref0re a110wed exam1nat10n 0f the dec11ne 1n 5CE5 t0 c0ntr01 1eve15 1n the treated 9r0up. 8a5e11ne 5CE fre4uency 1n therapeut1ca11y treated pat1ent5 dec11ned t0 the fre4uency 0f c0ntr01 pat1ent5 (5.25 5CE/ce11) w1th1n 2-5 m0nth5. 1t 15 1ntere5t1n9 t0 n0te that the dec11ne 0f PM-1nduced 5CE 1eve15 appear5 51m11ar t0 the dec11ne 0f the 6a5e11ne 5CE 1eve15 (5ee F19. 1). 5tat15t1ca1 exam1nat10n 0f the 6eta c0eff1c1ent5 0f the three curve5 revea15 n0 d1fference 1n rate 0f dec11ne. 7a61e 3 d15p1ay5 Pear50n c0rre1at10n5 6etween 6a5e11ne, 1nduced 5CE 5c0re5 and the num6er 0f m0nth5 51nce the 1a5t treatment d05e w1th 6a5e11ne and net 1nduced 5CE 5c0re5. Net 1nduced 5CE 5c0re5 are va1ue5 06ta1ned 6y 5u6tract1n9 the 6a5e11ne 5CE va1ue fr0m PM-1nduced 5CE 1eve15. Pear50n•5 c0rre1at10n c0eff1c1ent mea5ure5 the 5tren9th 0f a550c1at10n 6etween tw0 c0nt1nu0u5 var1a61e5 (5nedec0r, 1976). 8a5e11ne 5CE 5c0re5
276 7A8LE 3 P E A R 5 0 N C 0 R R E L A 7 1 0 N 5 0 F 71ME 51NCE LA57 D 0 5 E 8A5EL1NE A N D C H A L L E N 6 E 5CE 5 C 0 R E (n)
(1) 71me 51nce1a5t 1n-v1v0d05e(m05.) (2) 8a5e11ne5CE 5c0re (3) 1nduced0.1 #9/m1 PM (4) 1nduced0.25 #9/m1 PM
8a5e11ne
Net a 0.1 #9/m1 PM
Net 6 0.25 #9/m1 PM
-0.55 (19) *
-0.48 (19) *
-0.01 (19)
-
0.05 (39) -
-0.19 (39) - 0.58 (39) * * -
* p < 0.05. ** p < 0.0001. a 1nduced 0.1 #9/m1 PM 5CE 1eve1 m1nu5 6a5e11ne 5CE 1eve1. 6 1nduced 0.25 #9/m1 PM 5CE 1eve1 m1nu5 6a5e11ne 5CE 1eve1.
are n0t c0rre1ated w1th net 1nduced 5CE 5c0re5 at e1ther the 0.1 #9/m1 PM cha11en9e ( r = 0.05; n0t 519n1f1cant (N5)) 0r at the 0.25 #9/m1 PM cha11en9e ( r = - 0 . 1 9 ; N5). H0wev0r 5CE 5c0re5 f0r the tw0 cha11en9e5 are h19h1y c0rre1ated ( r = - 0 . 5 8 ; p < 0.0001). 7he num6er 0f m0nth5 51nce the 1a5t therapeut1c chem0therapy d05e 5h0wed a 5tr0n9 ne9at1ve c0rre1at10n w1th 6a5e11ne 5CE 5c0re5 (r = - 0 . 5 5 ; p < 0.05) and at the 10w d05e cha11en9e ( r = - 0 . 4 8 ; p < 0.05). 51m11ar re5u1t5 were 06ta1ned u51n9 5pearman•5 rh0 and w1th 109-tran5f0rmed data. D15cu5510n 1ncrea5ed fre4uency 0f 6a5e11ne 5CE5 1n P 8 L 0f pat1ent5 rece1v1n9 ant1cancer therapy ha5 6een rep0rted prev10u51y (Lam6ert et a1., 1978; Vya5 et a1., 1988; Pa1mer et a1., 1984). 0 u r data 5h0w1n9 a 32% d1fference 6etween 6a5e11ne 5CE5 1n the 9r0up therapeut1ca11y treated w1th CP c0mpared t0 untreated c0ntr015, 15 c0mpara61e t0 the5e prev10u5 rep0rt5. A1th0u9h therapeut1ca11y treated pat1ent5 had 1ncrea5ed 6a5e11ne 5CE fre4uenc1e5 c0mpared t0 c0ntr01 pat1ent5, the net 1ncrea5e 1n PM-1nduced 5CE5 1n the therapeut1ca11y treated pat1ent5 wa5 n0t 9reater than 1n c0ntr01 pat1ent5. 1ndependent var1a61e5 w1th p0tent1a1 1nf1uence 0n 5CE fre4uency (5m0k1n9, j06 h15t0ry, a1c0h01 u5e) were f0und n0t t0 have 519n1f1cant effect e1ther 0n 6a5e11ne 0r PM-1nduced 1eve15. 7he5e re5u1t5 are perhap5 n0t 5urpr151n9 91ven the 5ma11 num6er5 0f 5u6ject5 wh0 current1y 5m0ke 0r w0rk
1n a ha2ard0u5 0ccupat10n. Prev10u5 therapeut1c treatment w1th CP wa5 a 519n1f1cant effect0r 0f 5CE 1eve15 when exam1n1n9 ever-ver5u5-never treated, a1th0u9h 11fet1me therapeut1c CP d05e d1d n0t c0rre1ate w1th 5CE 1eve15, perhap5 6ecau5e 0f the re1at1ve1y rap1d 5CE dec11ne. 7he effect 0f t1me 51nce 1a5t therapeut1c treatment 0n 5CE 1eve15 wa5 exam1ned a5 5een 1n F19. 1. 8a5e11ne and PM-1nduced 5CE 1eve15 1n therapeut1caUy treated pat1ent5 were 5tr0n91y 1nf1uenced 6y the t1me 51nce 1a5t chem0therapy treatment. 8a5e11ne 5CE 1eve15 1n CP-treated pat1ent5 have 6een rep0rted t0 return t0 n0rma1 5-6 week5 after the 1a5t treatment (Rap05a, 1986). Re5u1t5 fr0m the pre5ent 5tudy 1nd1cate that cyc10ph05pham1de-1nduced 5CE5 were f0und t0 dec11ne t0 n0rma1 1n appr0x. 4 - 6 week5 re9ard1e55 0f dru9 d05e rece1ved. 7he dec11ne 1n 5CE5 c0u1d re5u1t fr0m repa1r 0f cyc10ph05pham1de-1nduced 1e510n5, rep1acement 0f dama9ed 1ymph0cyte5 fr0m a p001 0f undama9ed precur50r5 0r a c0m61nat10n 0f the5e tw0 pr0ce55e5. 1n v1tr0 114u1d h01d1n9 5tud1e5 have 5h0wn that 60-arre5ted human 1ymph0cyte5 exh161t d1fferent1a1 repa1r 0f D N A 1e510n5 1nduced 6y d1fferent c1a55e5 0f muta9en1c a9ent5 (L1tt1ef1e1d et a1., 1979). A150 rep0rted ha5 6een a 510wer n0rma112at10n rate when CP wa5 91ven 1n a c0m61nat10n re91me wh1ch 1nc1uded pr0car6a21ne 1n repet1t1ve cyc1e5 (Rap05a and Vark0ny1, 1987). 7h15 may repre5ent the added 9en0t0x1c1ty 0f pr0car6a21ne, 0r the 1na6111ty 0f DNA-repa1r mechan15m5 t0 dea1 w1th 5ucce551ve r0und5 0f cyt0t0x1c chem0therapy. Rap05a and 0ther5 have dem0n5trated that cyt0-
277
5tat1c dru95 act1n9 d1rect1y 0n DNA (a1ky1at1n9 a9ent5, 1nterca1at1n9 a9ent5) 1nduce 5CE, wherea5 a9ent5 5uch a5 5p1nd1e p0150n5 (v1nca a1ka101d5) and the ant1meta6011te5 (5-f1u0r0urac11, meth0trexate) d0 n0t 1nduce 0r have 11tt1e effect 0n 5CE5 (W01ff, 1982; Rap05a, 1978; Rap05a and Vark0ny1, 1987; 0ht5uru et a1., 1980). 7h15 065ervat10n 5u99e5t5 that a chem0therapy re91me 1nc1ud1n9 mu1t1p1e r0und5 0f therapy w1th 5CE 1nduc1n9 a9ent5 may re5u1t 1n a pr0tracted return t0 n0rma1 5CE 1eve15. 5tud1e5 1n exper1menta1 an1ma15 a150 5u99e5t that the fre4uency 0f exp05ure affect5 the rate 0f dec11ne f0r muta9en-1nduced 5CE5. F0r examp1e, 5tetka et a1. rep0rted that f0110w1n9 a 51n91e 1nject10n 0f m1t0myc1n C, 5CE 1eve15 1n ra661t per1phera1 1ymph0cyte5 returned t0 n0rma1 w1th1n 1 week. After exp05ure t0 mu1t1p1e d05e5, 5CE 1eve15 per515ted f0r at 1ea5t 4 week5 f0110w1n9 the f1na1 1nject10n. 7he auth0r5 rep0rted that the effect c0u1d 6e a5cr16ed t0 the appearance and per515tence 0f a 5u6p0pu1at10n 0f ce115w1th h19h 5CE va1ue5 (5tetka et a1., 1979). We a150 1nve5t19ated the p05516111ty0f u5a9e 0f mu1t1p1e 9en0t0x1c a9ent5 pr0tract1n9 the return t0 n0rma1 5CE 1eve15 1n 0ur therapeut1ca11y treated 9r0up, 6y 5trat1fy1n9 pat1ent5 1nt0 therapy cate90r1e5 w1th 0n1y 0ne 5CE 1nducer 5uch a5 CP a10ne 0r 1n c0m61nat10n w1th an ant1-meta6011te 0r 5p1nd1e p0150n, ver5u5 a 9r0up1n9 w1th m0re than 0ne 5CE-1nduc1n9 a9ent 5uch a5 CP p1u5 adr1amyc1n, an 1nterca1at1n9 a9ent. N0 d1fference 1n the rate 0f 5CE dec11ne wa5 5een; perhap5 6ecau5e the 5trat1f1ed 9r0up5 were very 5ma11. 7he pre5ent 5tudy c0nf1rmed prev10u5 065ervat10n5 0f 519n1f1cant 1ncrea5e5 1n 6ack9r0und 5CE fre4uenc1e5 1n cancer pat1ent5 treated w1th a1ky1at1n9 ant1cancer dru95 when c0mpared t0 untreated c0ntr015. 7he net 1ncrea5e 1n PM-1nduced 5CE5 1n therapeut1ca11y treated pat1ent5, h0wever wa5 n0t 519n1f1cant1y d1fferent than 1ncrea5e5 1n untreated c0ntr01 pat1ent5. 71me 51nce 1a5t 1n-v1v0 chem0therapy treatment 5tr0n91y 1nf1uenced 60th 5p0ntane0u5 and PM-1nduced 5CE 1eve15, 1rre5pect1ve 0f t0ta1 dru9 d05e rece1ved. 1n add1t10n, the rate 0f dec11ne 1n 5CE e1evat10n5 d1d n0t d1ffer 6etween the 5p0ntane0u5 and PM-1nduced curve5. Further 5tudy 0f muta9en-1nduced 5CE 1eve15 are needed t0 e1uc1date the1r va1ue a5 p0tent1a1
1nd1cat0r5 0f prev10u5 human exp05ure. VarY1n9 the 5CE cha11en91n9 a9ent may revea1 c0mp0und5pec1f1c re1at10n5h1p5 6a5ed 0n prev10u5 muta9en exp05ure.
Ackn0w1ed9ement5 7h15 w0rk wa5 5upp0rted 1n part 6y the Me110n F0undat10n and D H H 5 6rant RR05445. 7he auth0r5 w15h t0 thank M5. Chandra 5taten f0r 5k111edmanu5cr1pt preparat10n.
Reference5 80chk0v, N.P., 7.V. F111pp0va, 5.M. Ku21n and 5.V. 5tuka10v (1986) Cyt09enet1c effect5 0f cyc10ph05pham1de 0n human 1ymph0cyte5 1n-v1v0 and 1n-v1tr0, Mutat10n Re5., 159, 103107. Cha6ner, 8.A. (1977) 5ec0nd ne0p1a5m - - a c0mp11cat10n 0f cancer chem0therapy, N. En91. J. Med., 297, 213-215. Dearf1e1d, K.L., D. Jac0650n-Kram, 5.K. 8uenaventura and J.R. W1111am5 (1985) 1n-v1tr0 a55ay5 0f 1n-v1v0 exp05ure t0 cyc10ph05pham1de: 1nduct10n 0f 515ter-chr0mat1d exchan9e5 1n per1phera1 1ymph0cyte5, 60ne marr0w ce115 and 1n cu1tured ce115 exp05ed t0 p1a5ma, Mutat10n Re5., 158, 92-104. Han5teen, 1.-L. (1982) 5CE a5 a m0n1t0r 0f 1ndu5tr1a1 and env1r0nmenta1 t0x1n5, 1n: A.A. 5annd6erd (Ed.), 515terchr0mat1d Exchan9e, Pr09re55 and 70p1c5 1n Cyt09enet1c5, 2nd edn., L155, New Y0rk, pp. 675-698. H00ver, R., and J.F. Franmen1 Jr. (1981) Dru9-1nduced cancer, Cancer, 47, 1071-1080. K1m, J.P., P. D•Arpa, D. Jac0650n-Kram and J.R. W1111am5 (1985) U1tra-v101et-119ht exp05ure 1nduce5 a her1ta61e 5en51t1v1ty t0 the 1nduct10n 0f 5CE 6y m1t0myc1n-C, Mutat10n Re5., 149, 437-442. Lam6ert, 8., Y. R1n960r9, E. Harper and A. L1nd61ad (1978) 515ter chr0mat1d exchan9e5 1n 1ymph0cyte cu1ture5 0f pat1ent5 rece1v1n9 chem0therapy f0r ma119nant d150rder5, Cancer 7reat. Rep., 62, 1413-1419. Latt, 5.A., J. A11en, 5.E. 8100m, A. Carran0, E. F01ke, D. Kram, E. 5chne1der, R. 5chreck, R. 71ce, 8. Wh1tf1e1d and 5. W01ff (1981) 515ter-chr0mat1d exchan9e5: a rep0rt 0f the 6ene-70x Pr09ram, Mutat10n Re5., 87, 17-62. L1tt1ef1e1d, L.6., 5.P. C01yer, A.M. 5ayer and R.J. DuFra1n (1979) 515ter-chr0mat1d exchan9e5 1n human 1ymph0eyte5 exp05ed dur1n9 6 0 t0 f0ur c1a55e5 0f DNA-dama91n9 chem1ca15, Mutat10n Re5., 67, 259-269. Lund9ren, K., and 6.W. Luc1er (1985) D1fferent1a1 enhancement 0f 515ter-chr0mat1d exchan9e fre4uenc1e5 6y a-naphth0f1av0ne 1n cu1tured 1ymph0eyte5 fr0m 5m0ker5 and n0n-5m0ker5, Mutat10n Re5., 216, 307-308. Me1Y1n9, C., and 2. X1ant1n9 (1982) C0mparat1ve 5tud1e5 0n 5p0ntane0u5 and m1t0myc1n C-1nduced 515ter chr0maf1d exchan9e5 1n 5m0ker5 and n0n-5m0ker5, Mutat10n Re5., 105, 195-200.
278 Natarajan, A.7., and L.H.F. Mu11ender5 (1987) 515ter-chr0mat1d exchan9e, 1n: 6. 06e and A. 8a51er (Ed5.), Cyt09enet1c5, 5pr1n9er, New Y0rk, pp. 338-344. 0ht5uru, M., Y. 15h11,H. 1-119a5k1and 6. Karak1 (1980) 515terchr0mat1d exchan9e 1n 1ymph0cyte5 0f cancer pat1ent5 rece1v1n9 m1t0myc1n C treatment, Cancer Re5., 40, 477-480. Pa1mer, 1L6., C.F. 1)0re and A. M1chae1 Denman (1984) Ch10ram6uc11-1nduced chr0m050me dama9e t0 human 1ymph0cyte5 15 d05e-dependent and cumu1at1ve, Lancet, 1, 246-249. Rap05a, 7. (1978) 515ter-chr0mat1d exchan9e 5tud1e5 f0r m0n1t0r1n9 DNA dama9e and repa1r capac1ty after cyt05tat15t1c5 1n-v1tr0 and 1n 1ymph0cyte5 0f 1eukem1c pat1ent5 under cyt05tat1c therapy, Mutat10n Re5., 57, 241-251. Rap05a, 7. (1984) 5CE 1nduct10n 6y cyt05tat1c5 and 1t5 re1at10n t0 1atr09en1c 1eukem09ene515, 8a51c L1fe 5c1., 29, 859-884. Rap05a, 7., and J. Vark0ny1 (1987) 7he re1at10n5h1p 6etween 515ter chr0mat1d exchan9e 1nduct10n and 1enkem09en1c1ty, Cancer Det. Prev., 106, 141-151. 5nedec0r, 6e0r9e W., and W1111am6. C0chran (1976) 5tat15t1-
ca/ Meth0d5, 6th edn., 10wa 5tate Un1ver51tyPre55, Ame5, 1A, pp. 172-198. 50r5a, M., and J.W. Ya9er (1987) CYt09enet1c 5urve111ance0f 0ccupat10na1 exp05ure5, 1n: 6. 06e and A. 8a51er (Ed5.), CYt09enet1c5, 5pr1n9er, New Y0rk, pp. 345-360. 5tetka, D.6., J. M1n1der and A.V. Carran0 (1979) 1nduct10n 0f 10n9-11ved chr0m050me dama9e a5 man1fe5ted 6y 515ter chr0mat1d exchan9e 1n 1ymph0cyte5 0f an1ma15 exp05ed t0 m1t0myc1n-C, Mutat10n Re5., 51, 383-396. 5t011ey, P.D., K.A. 50per, 5.M. 6a110way, W.W. N1ch015, 5.A. N0rman and 5.R. W01man (1984) 515ter-chr0mat1d exchan9e5 1n a550c1at10nw1th 0ccupat10na1 exp05ure t0 ethy1ene 0x1de, Mutat10n Re5., 129, 89-102. Vya5, R.C., 5.6. Adhvaryu and V.C. 5hah (1988) Effect5 0f CCNU therapy 0n human chr0m050me5, Mutat10n Re5., 206, 163-166. W01ff, 5. (1982) D1ff1cu1t1e5 1n a55e551n9 the haman hea1th effect5 0f muta9en carc1n09en5 6y cyt09enet1c ana1y5e5•, Cyt09enet. Ce11. 6enet., 33, 7-13.
