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The effect of agent dose and treatment time on the intercellular distribution of sister-chromatid exchanges induced by genotoxic agents in mouse bone marrow cells in vivo
Mutation Research, 215 (1989) 25-37 Elsevier
25
MUT 04800
The effect of agent dose and treatment time on the intercellular distribution of sister-chromatid exchanges induced by genotoxic agents in mouse bone marrow cells in vivo Raymond R. Tice a Brian G. Ormiston a,. and Alfred F. McFee b a Medical Department, Brookhaven National Laboratory, Upton, N Y 11973 and 6 Medical Sciences Division, Oak Ridge Associated Universities, P.O. Box 117, Oak Ridge, TN 37831 (U.S.A.)
(Received 13 April 1989) (Revision received 7 June 1989) (Accepted 13 June 1989)
Keywords: Sister-chromatid exchanges; Dispersion analysis; Mouse bone marrow; Mitomycin C; Cyclophosphamide; Dimethylbenzanthracene
Summary Using two methods of bromodeoxyuridine (BrdUrd) administration and three genotoxic chemicals, the effects of dose and treatment time on the intercellular distribution of sister-chromatid exchanges (SCE) in the bone marrow of male B6C3F1 mice were evaluated. The dispersion of SCE among solvent control mice infused intravenously with BrdUrd or implanted subcutaneously with a BrdUrd tablet partially coated with paraffin was largely consistent with a Poisson model. Intraperitoneal treatment with cyclophosphamide (CP; solvent = phosphate-buffered saline), 7,12-dimethylbenzanthracene (DMBA; solvent = corn oil) and, in mice infused with BrdUrd, mitomycin C (MMC; solvent = phosphate-buffered saline) induced a significant increase in SCE, the distribution of which was not distributed as a Poisson. For CP and MMC, the increase in dispersion was dose-dependent and independent of treatment time ( - 1 , + 1 or + 8 h in relation to the start of the BrdUrd treatment). The lack of a treatment time effect suggests that there were no significant differences among treatment times in the distribution of the reactive forms of these two chemicals, no variation in cell-stage sensitivity, and no cellular toxicity to modulate the response. For DMBA, the increased dispersion of induced SCE depended on treatment time and was not simply related to dose. The increase in dispersion was agent-specific; at equal levels of SCE induction, the distribution of SCE in mice treated with DMBA exhibited greater dispersion than SCE in mice treated with either CP or MMC. These differences between DMBA and C P / M M C are probably due to DMBA's slower absorption/distribut!on kinetics, its requirement for metabolic activation to genotoxic metabolites and its extended half-life. These data suggest that analyzing the distribution of SCE, in addition to mean frequency, is a useful method for evaluating agent specific patterns in SCE induction.
Present address: Environmental Services Division, Reynolds, Smith and Hills, Inc., P.O. Box 22003, Tampa, FL 33622
(U.S.A.).
Correspondence: Dr. Raymond Tice, Integrated Laboratory Systems, P.O. Box 13501, Research Triangle Park, NC 27709 (U.S.A.).
0027-5107/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
26 The observed frequency of sister-chromatid exchanges (SCE) by a genotoxic chemical depends on the molecular target of the inducing agent (Perry and Evans, 1975; Littlefield, 1982; Takehisa, 1982), the extent of lesion persistence (Stetka et al., 1978; Sasaki et al., 1982; Shafer, 1982; Tice and Schvartzman, 1982), the temporal relationship between the formation of the DNA damage and the onset of scheduled DNA synthesis (Stetka et al., 1978; Schvartzman and Gutierrez, 1980; Sasaki, 1982; Shafer, 1982), and the extent of cell lethality associated with the treatment. The observed frequency of SCE in vivo also depends on various pharmacokinetic considerations (e.g., route of exposure, reactive species half-life) and intrinsic factors such as species, strain, sex, age of the animal (Tice et al., 1982), and the target tissue (Conner et al., 1979). Recently, we investigated the effect of treatment time in relation to the onset of BrdUrd exposure on the magnitude of the SCE response generated by treating mice with one of three well-known genotoxic chemicals - c y c l o p h o s p h a m i d e (CP), 7,12-dimethylbenzanthracene (DMBA) and mitomycin C (MMC) (Tice et al., 1987). In that study, the effect of the method of bromodeoxyuridine (BrdUrd) administration (i.e., intravenous infusion versus a paraffin-coated tablet implanted subcutaneously) on the induction of SCE by MMC, as well as interlaboratory differences in response, were examined. We found that the induction of SCE, as measured by individual animal mean SCE frequency, was independent of the method of BrdUrd administration and independent of (for MMC) or dependent on (for CP and DMBA) treatment time (Tice et al., 1987). In 1982, Rainaldi and Mariani published a paper in which they discussed the importance of evaluating the intercellular dispersion of in vitro SCE data as a method for discriminating among agents with differing mechanisms of action. More recently, Margolin and Shelby (1985) demonstrated that groups of individuals with nonsignificantly different SCE means could have significantly different SCE/cell dispersions. These studies prompted us to re-evaluate the SCE data obtained in the in vivo mouse bone marrow treatment time study for the effect of chemical agent, treatment time and method of BrdUrd administration on the intercellular distribution of SCE within
individual animals. We anticipated that this analysis would provide information on the distribution of SCE among cells in control mice, as well as insight into the intercellular distribution of DNA damage induced by three different genotoxic chemicals. Materials and methods
A complete description of the materials and methods used in this study can be found in Tice et al. (1987). Male B6C3F1 mice (Frederick Cancer Research Center, Frederick, MD), between 10 and 14 weeks of age, were used. Food (Purina Laboratory Chow 5001) and water were provided ad libitum. Mitomycin C (CAS No. 50-07-7), BrdUrd, and reagent-grade corn oil were purchased from Sigma; colchicine from Eli Lilly, CP (CAS No. 50-18-0; Cytoxan) from Mead Johnson, DMBA (CAS No. 57-97-6) from Eastman Kodak, Hoechst 33258 from American Hoechst, Giemsa from Harleco. MMC and CP were dissolved in phosphate-buffered saline (PBS; pH 7.4); DMBA was suspended in corn oil. All injections were intraperitoneal (i.p.) at a volume of 0.1 ml (MMC) or 0.2 ml (CP, DMBA). In some experiments conducted at Brookhaven National Laboratory (BNL), individually restrained mice were continuously infused intravenously (i.v.) for 24 h with BrdUrd (50 m g / k g / h ) in PBS, while in other experiments at BNL and at Oak Ridge Associated Universities (ORAU), a 50 mg BrdUrd tablet, partially coated (70%) with paraffin (McFee et al., 1983) was implanted subcutaneously (s.c.). At 1 h before, 1 h after, or 8 h after the beginning of exposure to BrdUrd, groups of animals were injected i.p. with one of the three chemicals or the appropriate solvent. 20-22 h after the beginning of BrdUrd exposure, each animal was injected with colchicine. 2 h later, the mice were killed by cervical dislocation, femoral bone marrow was removed, exposed to hypotonic solution and fixed using 3 : 1 methanol : glacial acetic acid. Coded slides were prepared, differentially stained and 25 second-generation metaphase cells per animal were analyzed for SCE.
Statistical analysis. For all statistical analyses, significance was determined at an alpha level of
27 0.05. T o c o m p a r e the effects of dose and treatm e n t time o n the intercellular d i s t r i b u t i o n of S C E within individual animals, the dispersion coefficient H ( S n e d e c o r and C o c h r a n , 1967) w h i c h is the ratio of the s a m p l e variance to the s a m p l e m e a n ( M a r g o l i n an d Shelby, 1985) was analyzed by two - way A N O V A . T o d e t e r m i n e w h e t h e r the distribution of S C E within individual animals fit a Poisson model, the dispersion test ( S n e d e c o r a n d C o c h r a n , 1967) was applied. I n this a p p r o a c h , the statistic T = ( r - 1 ) H , w h e r e r is the n u m b e r of cells analyzed for SCEs, was calculated. T h e statistic T is referred to a table of critical values for the C h i - s q u a r e d i s t r i b u t i o n with r - 1 degrees of f r e e d o m ( M a r g o l i n et al., 1986). T o d e t e r m i n e w he the r the S C E f r e q u e n c y d i s t r i b u t i o n for groups of mice used as controls or treated with various doses of the car ci no g e n s fit a Poisson model, animals treated with the same dose were e x a m i n e d collectively by the dispersion test, c o m b i n i n g where a p p r o p r i a t e across t r e a t m e n t time groups. In addition, the co r r el at i o n b e t w e e n H and S C E
f r e q u e n c y was d e t e r m i n e d using linear regression analysis, c o m b i n i n g d a t a across t r e a t m e n t times w h er e a p p r o p r i a t e .
Results
Cyclophosphamide (BNL). T h e S C E d a t a for each animal, by dose an d t r e a t m e n t time, along with the i n d i v i d u a l m o u s e m e a n S C E f r e q u e n c y a n d the c a l c u l a t e d dispersion coefficient H are p r o v i d e d in T a b l e 1. C P at doses of 6.25, 12.5 an d 25.0 m g / k g i n d u c e d a linear increase in S C E f r e q u e n c y in mice i n f u sed with B r d U r d (Tice et al., 1987). T h e slope of the S C E response d e p e n d e d on t r e a t m e n t time. W h e n the f r e q u e n c y distribution of S C E d a t a within each c o n t r o l a n i m a l was analyzed b y the dispersion test for goodness of fit with a Poisson model, only 1 of 11 an i m al s differed significantly f r o m e x p e c t a t i o n s ( T a b l e 1). T r e a t m e n t with C P i n d u c e d a significant increase in the dispersion of S C E within i n d i v i d u a l animals ( P < 0.01) w h i ch was m a r g i n a l l y i n d e p e n d e n t of
TABLE 1 INDUCTION OF SCE BY CYCLOPHOSPHAMIDE Dose
-1 h a
(mg/kg)
Mean +_SEM b
0
+1 h a Range c
Hd
+8h a
Mean -+SEM b
Range c
Hd
Mean -+SEM b
Range c
Hd
1- 7 0- 8 2- 9 1- 14
0.75 1.18 0.82 1.49
4.00+0.38 4.00+_0.42 6.04+0.69 6.12+0.52
1- 7 1- 9 2-18 1-11
0.90 1.11 1.95 * 1.10
2.80-+0.28 3.96+0.39 7.20+0.42
0- 6 2- 8 4-11
0.71 0.94 0.63
12.48+0.64 16.44+1.00 16.80+_1.28 17.16_+0.86
7-19 10-31 6-33 9-24
0.82 1.53 * 2.44 * 1.07
13.40-+0.85 13.92-+0.80 16.12-+0.93 19.24_+1.32
6-27 8-22 7-26 7-36
1.34 1.16 1.35 2.26 *
22.20-+ 1.40 25.00-+1.26 25.36-+1.99 26.16_+1.33
815816-
34 40 47 40
2.20 1.58 3.90 1.69
12.5
21.08+0.98 26.44+1.10 29.76+_1.50 31.64+_2.22
12-30 15-38 17-49 4-51
1.13 1.13 1.89 * 3.90 *
24.56-+1.65 28.96+2.27 29.88+1.66 38.20+1.95
12-44 13-62 20-51 21-59
2.78 4.44 2.30 2.48
* * * *
36.24+1.88 36.68 + 3.01 39.68+2.03 47.00-+1.67
2291933-
58 79 64 64
2.45 * 5.86 * 2.60 * 1.49
25.0
41.16+-3.90 46.16 +-2.20 49.32+_2.80 59.52+3.19
13-85 29-70 22-73 32-89
9.25 2.63 3.96 4.27
50.00+-2.76 50.60 +-3.09 51.96+_1.66 65.28+_2.43
28-84 23-81 35-69 44-93
3.80 * 4.72 * 1.33 2.25 *
55.80 + 2.49 61.00+2.89 61.20+3.58 75.44+2.84
37- 84 31- 84 28- 92 48-109
6.25
* * * *
a Treatment time in relation to the onset of exposure to BrdUrd. b Animal mean SCE/cell frequency 5: standard error of the mean among 25 cells. Range of SCE values. d Dispersion coefficient = variance/mean. * Significantly different at a = 0.05 from Poisson distribution.
2.96+0.30 4.08_+0.44 5.04+0.41 7.08+0.65
2.77 3.43 5.23 2.67
* * * *
* * * *
28 TABLE 2 DI SPERSION ANALYSIS OF C P - I N D U C E D SCE DATA
/ /
Dose (mg/kg)
Nu mb er of animals
Number ofexceptions ~
Sum b X2
df ~
/
Critical J X2
0
11
1
277.8
264
302.6
6.25
12
7
646.6 *
288
328.3
12.50
12
9
778.7 •
288
328.3
25.0
12
11
1119.9 •
288
328.3
//I / •
treatment time ( P = 0.06). A dispersion analysis of SCE data from mice at each dose combined across treatment times indicated that while the 'I
•
l--
I.
•
•
II
AOA
.
•
/
J
i .I ~-
Data combined for treatment times - 1 , + 1, + 8 h. Number of animals whose SCE data were significantly different from Poisson expectations. h Sum of Chi square values. ~ Degrees of freedom. d Critical Chi square value for a = 0.05. * Significantly different at a = 0.05.
tD
/
•
i A
/Oo- : i
o
SCE FREQUENCY
Fig. 2. Plot of the correlation between SCE frequency and the dispersion correlation H for dimethylbenzanthracene. Each data point represents the H value and SCE frequency for an individual mouse basedon an analysisof 25 second-generation metaphase cells. Square ~ - l h treatment time data. Linear regression analysis of the data indicated a significant regression of H against SCE frequency (regression coefficient r = 0.6059; P = 0.0167; y ~ 0.917 +0.096 x denoted by the solid line); Circle = + 1 h treatment time data. Linear regression analysis of the data indicated a significant regression of H against SCE frequency (regression coefficient r = 0.7207; P = 0.0007; y = 0.415+0.084 x denoted by the dashed line); Triangle = + 8 h treatment time data. Linear regression analysis of the data indicated a significant regression of H against SCE frequency (regression coefficient r = 0.7859; P = 0.0003; y = 0.005 +0.169 x denoted by the dash-dotted line). Solid symbols indicate SCE distributions for an individual mouse significantly different from Poisson expectations at c~= 0.05.
8o
data for control animals fit a Poisson model, the distribution in CP-treated mice differed significantly from Poisson expectations at each dose (Table 2). With increasing dose, the SCE response became increasingly hyperdispersed (Fig. 1).
Fig. 1. Plot of the correlation between SCE frequency and the dispersion correlation H for cyclophosphamide. Each data point represents the H value and SCE frequency for an individual mouse based on an analysis of 25 second-generation metaphase cells. Square = - 1 h treatment time data; Circle = + 1 h treatment time data. Triangle = + 8 h treatment time data. Linear regression analysis of the data pooled across treatment times indicated a significant regression of H against SCE frequency (regression coefficient r = 0.5724; P < 0.0001; y =1. 015+0.0 48 x denoted by the solid line). Solid symbols indicate SCE distributions for an individual mouse significantly different from Poisson expectations at a = 0.05.
Dimethylbenzanthracene (ORAU). The individual cell SCE data, along with each animal's mean frequency and the calculated H statistic, for mice treated with DMBA at doses of 0, 10, 50 and 300 m g / k g at - 1 , + 1 and + 8 h relative to the time of BrdUrd tablet implantation can be found in Table 3. The shape and magnitude of the SCE dose-response for DMBA was highly dependent upon treatment time (Tice et al., 1987). With decreasing time between treatment and sampling
i•
o
L
,o
•
2'0
3'o
4'0
•
•
i
5o
•
m
6o
;o
8CE FREQUENCY
29 TABLE 3 INDUCTION
OF SCE BY 7,12-DIMETHYLBENZANTHRACENE
Dose
-1 h a
(mg/kg)
Mean ± SEM b
Range ~
H d
M e a n -+ S E M b
0
3.96+_0.40 4.00-+0.49 6.72_+0.85
0-11 1- 8 0-16
1.53 * 0.98 2.71 *
10
11.60_+0.85 13.72_+1.24 14.92_+1.17 15.08_+1.43
4-20 5-30 7-27 5-31
1.55 2.80 2.28 3.40
50
15.20+_1.02 15.44_+1.07 17.56_+1.10 19.96 + 1.08
100
26.16_+1.96 28.24_+1.96 29.64_+1.42 29.68 _+ 2.66
a b c d *
+8h
+1 h a Range c
H a
a
Mean + SEM b
Range c
H d
4.56 ± 0.42 4.84+0.51 4.92+0.56 5.16+0.44
1- 9 1-10 1-11 1- 9
0.95 1.34 1.61 * 0.93
4.28_+0.41 4.52_+0.46 4.60_+0.45 4.76_+ 0.39 5.12_+0.44
1- 9 0-10 1-11 0- 8 1-11
0.98 1.18 1.12 0.81 0.95
* * * *
11.76_+0.65 11.92_+0.79 13.96_+0.82 15.40+_1.16 15.48 -+ 0.94
7-20 5-21 7-25 7-30 8-27
0.91 1.32 1.19 2.19 • 1.42
10.16+_0.89 12.04+0.77 12.08 + 1.10 23.04+2.09
2-21 6-19 3-23 9-47
1.95 * 1.24 2.52 * 4.76 *
8-26 7-29 8-30 11-33
1.73 * 1.86 * 1.72 * 1.46
16.36-+0.84 17.20_+1.25 20.32_+1.27 21.68 _+ 1.45
9-27 9-31 10-34 8-36
1.07 2.28 • 1.98 • 2.42 •
10.88_+0.83 11.96+0.81 12.56_+0.79 13.24_+0.75
5-21 6-23 5-21 7-24
1.56 * 1.37 1.24 1.06
11-48 13-73 20-51 16-75
3.68 4.54 1.69 5.95
20.52_+0.82 22.32_+1.45 22.60_+1.37 25.96 + 2.06
12-29 9-36 11-39 14-56
0.82 2.37 • 2.09 • 4.10 •
10.44_+0.87 11.44_+0.91 12.56_+1.01 16.12_+1.51
3-19 4-20 4-23 8-32
1.83 1.80 2.04 3.53
* * * *
* * * *
T r e a t m e n t time in relation to the onset of exposure to BrdUrd. A n i m a l m e a n S C E / c e l l frequency +_ standard error of the m e a n a m o n g 25 cells. R a n g e of SCE values. D i s p e r s i o n coefficient = v a r i a n c e / m e a n . Significantly different at a = 0.05 from Poisson distribution.
times, the magnitude of the SCE response was diminished and the shape of the dose response increasingly curvilinear. Both D M B A dose ( P < 0.01) and treatment time ( P = 0.04) had a significant effect on the dispersion of SCE within individual animals. Pairwise comparisons among the 3 treatment times indicated that the injection of mice with D M B A 1 h prior to BrdUrd tablet implantation induced a greater dispersion in SCE than any other treatment time. The presence of a treatment time effect prevented the combining of data within doses across treatment times. A dispersion analysis of the individual dose by treatment time group data indicated that only the data for the + 1 h treatment time control group was Poisson distributed (Table 4). When H was regressed against SCE frequency for each animal treated with D M B A within each treatment time (Fig. 2), a complex relationship between the in-
duced SCE response and the SCE distribution became apparent.
Mitomycin C (ORAU). The SCE data for animals implanted with a BrdUrd tablet and treated with M M C are given in Table 5. In addition to treatment times of - 1 , + 1 and + 8 h in relation to BrdUrd tablet implantation, some mice were injected i.p. with M M C at the time of BrdUrd treatment (i.e., at time 0). The mean SCE response for M M C at all treatment times was linear over the dose range tested (1.0-4.0 m g / k g ) , and without a significant treatment time effect (Tice et al., 1987). Analysis of the individual mouse SCE distribution data for goodness of fit to a Poisson model revealed that, with increasing dose, an increasing number of animals yielded data which deviated significantly from Poisson expectations (Table 5). Analysis by two-way A N O V A indicated
30
that neither the treatment time ( P -- 0.49) nor the treatment ( P = 0.08) had a significant effect on the dispersion of SCE data within individual mice. Therefore, all animals were combined across treatment time and dose and collectively analyzed by the dispersion test. A significant difference from a Poisson model was obtained (Table 6). A plot of the H statistic against each animal's respective SCE data is presented in Fig. 3.
g •
•
o
Mitomycin C (BNL). The individual cell SCE data by treatment time, method of BrdUrd administration, dose, and animal are provided in Table 7. MMC at doses of 1.0, 2.0, 2.5 and 3.0 m g / k g
~0
6 DISPERSION DATA
OF
ANALYSIS
DMBA-INDUCED
SCE
(A) T r e a t m e n t time = - 1 h
0 10 50 300
~'
o
25
3b
i
4o
i
50
65
SCE FREQUENCY
TABLE 4
Dose (mg/kg)
0
2o
Number of
Number o f excep-
animals
tions
3 4 4 4
2 4 3 4
Sum X2 b
df c
Critical d X2
a
125.1 240.9 162.5 380.7
* * * *
72 96 96 96
92.8 119.9 119.9 119.9
df c
Critical a
Fig. 3. Plot o f the c o r r e l a t i o n b e t w e e n S C E f r e q u e n c y and the dispersion c o r r e l a t i o n H f o r m i t o m y c i n C ( O R A U ) . E a c h d a t a point represents the H v a l u e a n d S C E f r e q u e n c y for a n individual m o u s e b a s e d o n a n a n a l y s i s o f 25 s e c o n d - g e n e r a t i o n m e t a p h a s e cells. S q u a r e = - 1 h t r e a t m e n t t i m e d a t a ; D i a m o n d = 0 t r e a t m e n t t i m e d a t a ; Circle = + 1 h t r e a t m e n t t i m e d a t a ; Triangle = + 8 h treatment time data. Linear regression analysis of the d a t a p o o l e d a c r o s s t r e a t m e n t times did n o t result in a s i g n i f i c a n t r e g r e s s i o n o f H against S C E f r e q u e n c y (regression c o e f f i c i e n t r = 0.2068; P = 0.1039; y = 1 . 1 0 7 + 0 . 0 0 6 x den o t e d by the solid line). Solid s y m b o l s i n d i c a t e S C E distributions for a n i n d i v i d u a l m o u s e s i g n i f i c a n t l y d i f f e r e n t f r o m Poisson e x p e c t a t i o n s at a = 0.05.
(B) T r e a t m e n t time = + 1 h Dose (mg/kg)
0 10 50 300
Number of
Number of excep-
animals
tions a
5 5 4 4
0 1 3 3
Sum X2 b
X2 121.6 168.9 * 186.2 * 225.2 *
120 120 96 96
146.3 146.3 119.9 119.9
Sum X2 b
df ~
Critical d
(C) T r e a t m e n t t i m e = + 8 h Dose (mg/kg)
0 10 50 300
Number of
Number of excep-
animals
tions
4 4 4 4
1 3 1 4
X2
a
115.8 251.3 * 125.8 * 220.9 *
96 96 96 96
119.9 119.9 119.9 119.9
N u m b e r of a n i m a l s w h o s e S C E d a t a were s i g n i f i c a n t l y different f r o m P o i s s o n e x p e c t a t i o n s . b S u m of Chi s q u a r e values. L. D e g r e e s of f r e e d o m . d Critical C h i s q u a r e v a l u e for c~= 0.05. * S i g n i f i c a n t l y d i f f e r e n t at a = 0.05.
induced a linear increase in SCE frequency which was independent of treatment time and of the method of BrdUrd administration (Tice et al., 1987). Analyzing the dispersion of SCE in individual animals resulted in finding a number of animals in which the SCE response failed to agree with a Poisson model (Table 7). Analysis of H by A N O V A using dose and treatment time or dose and method of BrdUrd administration as factors indicated for mice infused with BrdUrd that treatment with M M C resulted in a significant increase in H ( P < 0.01) which was independent of treatment time ( P = 0.11). A dispersion analysis of mouse data pooled across treatment times indicated a significant departure from a Poisson model at every dose of MMC (Table 8A). In control mice implanted with BrdUrd tablets, SCE were not distributed as a Poisson ( P < 0.01) and treatment with M M C marginally affected the dispersion of SCE (Table 8B). A graphic presentation
TABLE 5 I N D U C T I O N OF SCE BY M I T O M Y C I N C (ORAU) Dose
-lh
(mg/kg)
Mean -+ SEM b
a
Oh a Range c
H d
0
Mean + SEM b
+lh Range c
n d
4.96 + 0.47 5.76_+0.61 5.80+_0.48 6.20 ± 0.60
1- 9 2 13 1- 9 1-12
1.12 1.61 * 1.01 1.47
a
+8h a
Mean + SEM b
Range c
H a
Mean +_ SEM b
Range c
H d
1.0
15.80-+0.85 16.04-+0.72 16.32+0.95 17.00-+0.85
1010911-
28 25 29 31
1.14 0.80 1.39 1.07
11.32+0.73 14.52+0.86 18.44-+0.77 19.36-+1.11
7-22 6-24 13-26 10-32
1.18 1.26 0.81 1.58 *
14.28+0.84 16.28-+1.11 17.764-0.85 18.32_+0.95
9-28 6-28 11 27 11-30
1.23 1.90 * 1.02 1.23
15.60-t-0.56 15.88 -+ 0.90 16.00--+0.93 16.08 ± 0.72
11-20 9-27 8-28 9-22
0.50 1.27 1.36 0.80
2.5
16.24-t-0.61 19.60_+0.68 19.68-+0.95 24.80_+0.99
1012918-
21 28 31 37
0.58 0.58 1.14 0.98
15.88-+1.08 23.36+1.12 24.96+0.93 25.72_+1.03
9-29 12-40 17-39 17-36
1.82 * 1.34 0.87 1.04
25.04-+0.95 33.84-+1.23 39.60 + 1.35 41.80-+ 1.83
14-34 23-48 29-61 26-62
0.90 1.12 1.14 2.00 *
23.56_+0.95 24.764-0.99 39.56-+ 1.18 39.60 + 1.29
18-39 17-38 29-53 29-53
0.95 0.99 0.88 1.04
4.0
36.96+1.76 43.24_+1.69 49.92-+1.65 53.00_+3.01
22- 58 3 1 - 60 35- 71 36-110
2.10 * 1.64 * 1.37 4.27 *
41.08_+1.48 42.84_+1.28 49.60_+1.48
26-53 29-58 36-65
1.33 0.96 1.11
44.36-+1.19 50.52 + 1.51 51.04 + 1.76 59.64+ 1.65
30-55 37-65 37-73 48-76
0.80 1.13 1.52 * 1.15
41.32 + 1.35 49.56 + 1.35 50.52 -+ 1.51 54.68-+2.02
29-53 38-67 37-65 36-77
1.09 1.24 1.13 1.86 *
a b c d *
Treatment time in relation to the onset of exposure to BrdUrd. Animal mean S C E / c e l l frequency+ standard error of the mean a m o n g 25 cells. Range of SCE values. Dispersion coefficient = v a r i a n c e / m e a n . Significantly different at a = 0.05 from Poisson distribution.
32 TABLE 6 DISPERSION A N A L Y S I S OF M M C - I N D U C E D SCE DATA
(ORAU) Number of animals
Number of exceptions a
Sum b X2
df c
Critical o X2
52
10
1568.8 *
1248
1331.0
Data combined across doses and across treatment times of - 1 , 0 , +1, + 8 h . " Number of animals whose SCE data were significantly different from Poisson expectations. b Sum of Chi square values. c Degrees of freedom. d Critical Chi square value for a = 0.05. * Significantly different at a = 0.05.
tion of SCE in control mice injected with BrdUrd for 9 consecutive hours was consistent with Poisson expectations. At BNL, the majority of the mice were i.v. infused with BrdUrd dissolved in PBS, a limited number of mice were implanted s.c. with a BrdUrd tablet partially coated with paraffin. All control mice were injected i.p. with PBS, the solvent for MMC and CP. Among the 28 BrdUrd infused, solvent control mice, only 2 mice provided SCE data which were inconsistent with a Poisson model and analyzing the BrdUrd infused control animals by the dispersion test indicated that the pooled SCE data were distributed as a Poisson (Table 9).
of the relationships between H and SCE frequency for MMC can be found in Fig. 4. 4
Discussion
In vitro, the intercellular distribution of SCE within individual control culture dishes of V79 cells (Rainaldi and Mariani, 1982) or of CHO cells (Margolin et al., 1986), both functionally homogeneous populations, can be satisfactorily described by a Poisson model. In human peripheral blood lymphocytes mitogenically stimulated to proliferate in vitro, a Poisson distribution is inadequate for modelling the dispersion of SCE data within some individuals (Yakovenko and Platonova, 1979; Wulf et al., 1984; Whorton et al., 1984; Moore and Carraro, 1984; Sinha et al., 1985; Margolin and Shelby, 1985). This lack of fit suggests heterogeneity in sensitivity to BrdUrd among subpopulations of responding cells (Santesson et al., 1979; Crossen et al., 1986) a n d / o r prior exposure of the donor to genotoxic agents (Moore and Carrano, 1984). The latter explanation may be more likely because in a study involving mitogenically-stimulated rabbit lymphocytes, a dispersion analysis confirmed the adequacy of the Poisson model for describing the distribution of baseline SCE (DuFrain, 1983). Since bone marrow is comprised of several functionally distinct cell populations, it might be anticipated that the intercellular distribution of SCE values would be inconsistent with a simple Poisson model. However, Conner et al. (1978) concluded that the intercellular distribu-
3, ~ ~g~
~V , ,
0
Q
m
A
•
A
.
&
0
q)
,6>
•
z~
0
o
•
G
2'0
.~
4~
~
6~
8CE FREQUENCY
Fig. 4. Plot of the correlation between SCE frequency and the dispersion correlation H for mitomycin C (BNL). Each data point represents the H value and SCE frequency for an individual mouse based on an analysis of 25 second-generation metaphase cells. Square = - 1 h treatment time data for BrdUrd-infused mice; Circle = + 1 h treatment time data for BrdUrd-infused mice; Triangle = + 8 h treatment time for BrdUrd-infused mice. Linear regression analysis of the data pooled across treatment times indicated a significant regression of H against SCE frequency (regression coefficient r = 0.4762; P < 0.0001; y = 0 . 8 6 8 + 0 . 0 2 3 x denoted by the solid line). Diamond = +1 h treatment time data based on BrdUrd tablet-implanted mice. Linear regression analysis of the data pooled across treatment times did not result in a significant regression of H against SCE frequency (regression coefficient r = 0 . 2 8 3 5 ; P = 0 . 2 8 7 2 ; ) , = 1 . 4 2 6 + 0 . 0 1 0 x denoted by the dashed line). Solid symbols indicate SCE distribution for an individual mouse significantly different from Poisson expectations at a = 0.05.
a
18.92-+1.00 19.80_+ 1.36 20.56-1-1.13 22.76+1.80 23.92+1.22
24.80_+1.57 25.56+1.93 25.88 + 0.71 29.40-+ 1.52 35.64 + 0.97
34.48 + 1.63 36.36+1.72 42.28 5:1.34 53.00-+2.07 58.16+2.27
2.0
2.5
3.0
19-49 21- 5 6 31-60 34- 7 2 38-83
2-42 9-54 1 9- 34 13-53 28-45
11-28 9- 3 3 10-32 3-40 13-39
5-21 5-20 6-23 5-22 7-19
1- 7 2- 7 2- 9 1- 8 1-13 2-10 2-12
Range c
* * * *
1.93 2.04 1.06 2.02 2.21 * *
* *
2.48 * 3.64 * 0.49 1.96 * 0.66
1.32 2.33 1.56 3.55 1.55
1.50 1.32 1.15 1.23 1.18
0.67 0.47 0.92 0.93 1.84 * 0.97 0.84
H d
a
37.96 _+1.33 38.44_+1.62 47.92 -+ 2.32 53.36_+2.13
26.40_+1.09 27.84_+1.15 29.92 + 1.33 30.68 -+ 1.38 31.00-+ 0.87
19.52_+1.11 22.48 + 1.43 22.92-+0.86 23.56+1.10 24.92_+1.35
13.64-+0.66 3.84-+0.53 14.04 _+0.61 14.88 5:0.53 16.68-+0.81
3.96-+0.42 4 .08+ 0.29 4.20-+0.29 4.44+0.42 4.56_+0.44
Mean _+SEM b
+lh
9 7 7 9 9
25-52 25-59 30-82 40-82
18-37 13-36 18-47 20-49 23-39
12-32 12-36 14-30 15-36 15-43
8-22 10-20 9-21 10-21 9-24
02111-
Range ¢
1.17 1.72 * 2.81 * 2.12 *
1.13 1.19 1.48 1.56 * 0.61
1.58 * 2.28 * 0.81 1.28 1.83 *
0.80 0.52 0.66 0.48 0.97
1.13 0.51 0.52 1.00 1.06
H a
Treatment time in relation to the onset of exposure to BrdUrd. A ni m al mean S C E / c e l l frequency+ s ta nda rd error of the mean a m o n g 25 cells. Range of SCE values. Dispersion coefficient = variance~mean. Significantly different at a = 0.05 from Poisson distribution.
11.44+0.83 11.72+0.79 12.04 + 0.74 12.12 + 0.77 12.72:t:0.78
1.0
a b c a *
3.76+0.27 3.92-+0.32 4.32+0.40 4.64+0.42 5.445:0.63 6.08 + 0.49 6.72-1-0.47
Mean + SEM b
-lh
BrdUrd infusion
0
Dose (mg/kg)
I N D U C T I O N OF SCE BY M I T O M Y C I N C (BNL)
TABLE 7
40.52 _+ 1.33 41.40 + 1.94 45.56 + 2.25 45.76 -+ 1.34 48.80-+2.35
30.48 + 1.17 32.48 _+1.31 34.12 _+ 1.13 35.40 _+1.41 38.32_+1.39
25.68_+1.39 27.76 + 1.02 28.16 5:1.40 28.40 _+1.54 29.36+0.91
15.16+0.88 18.36_+1.14 18.48 _+ 1.09 19.52 _+0.96 21.40 __.0.91
3.76_+0.42 4.44+ 0.40 4.88 _+0.44 5.04_+0.43 5.64_+0.45
Mean + SEM b
+8h a
26-53 27-64 24-73 32-57 26-84
22-47 21-50 22-43 24-51 22-52
12-37 19-37 18-46 16-46 22-39
6-24 10-32 3-27 9-29 11-30
0- 8 1- 8 0- 9 1- 9 2-12
Range ¢
1.09 2.28 * 2.79 * 0.98 2.84 *
1.13 1.32 0.93 1.41 1.25
1.87 * 0.94 1.73 * 2.09 * 0.71
1.27 1.76 * 1.61 * 1.19 0.97
1.16 0.88 1.00 0.92 0.90
H a
a
33.68_+ 1.84 34.20 _+ 1.41 38.76 5:1.72 40.52 + 1.48
30.28 _+ 1.47 30.44 + 1.41 30.52_+1.43 36.16_+1.58
15.88_+0.85 18.32_+1.32 18.64_+1.31 18.68_+0.92
4.48+0.44 4.60+0.56 4.72+0.50 4.80+0.53
M ean -+ SEM b
+lh
Br d U r d tablet
19-54 18-47 24-55 26-52
15-47 21-43 17-54 26-53
9-23 6-33 6-35 11-28
1- 9 1-13 1-11 1 11
Range c
2.52 * 1.45 1.91 * 1.35
1.78 * 1.41 1.67 * 1.73 *
1.15 2.39 * 2.29 * 1.13
1.10 1.72 * 1.30 1.44
H a
34 TABLE 8 DISPERSION ANALYSIS OF MMC-INDUCED (BNL)
SCE DATA
(A) B r d U r d i.v. i n f u s i o n m e t h o d Dose (mg/kg)
0 1.0 2.0 2.5 3.0
Number of
Number o f excep-
animals
tions a
17 15 15 15 14
1 2 10 3 10
Sum b X2
df c
377.3 503.0 610.3 509.8 649.4
408 360 360 360 336
Critical d X2
* * * *
455.8 405.0 405.0 405.0 379.5
D a t a c o m b i n e d f o r t r e a t m e n t times - 1 , + 1, + 8 h. ~' N u m b e r o f a n i m a l s w h o s e S C E d a t a w e r e s i g n i f i c a n t l y different f r o m P o i s s o n e x p e c t a t i o n s . b S u m of C h i s q u a r e values. " Degrees of freedom. d Critical Chi s q u a r e value f o r a = 0.05. * S i g n i f i c a n t l y d i f f e r e n t at a = 0.05. (B) B r d t J r d t a b l e t m e t h o d Dose (mg/kg)
Number of animals
Number o f exceptions a
Sum b X2
0 1.0 2.5 3.5
4 4 4 4
1 2 3 2
133.4 167.0 158.l 173.5
h c d *
* * * *
Number of animals whose SCE data different from Poisson expectations. S u m of C h i s q u a r e values. D e g r e e s of f r e e d o m . Critical Chi s q u a r e v a l u e for a = 0.05. S i g n i f i c a n t l y d i f f e r e n t at a = 0.05.
df c
Critical d X2
96 96 96 96
119.9 119.9 119.9 119.9
were s i g n i f i c a n t l y
In mice at BNL implanted s.c. with a BrdUrd tablet, the SCE distribution was significantly hyperdispersed in 1 of 4 mice and the pooled group data were inconsistent with Poisson expectations (Table 9). This apparent difference between the intercellular distribution of SCE values within control mice depending on the method of BrdUrd administration occurred in spite of a lack of difference in the mean SCE frequency. At ORAU, all solvent control animals were implanted s.c. with a BrdUrd tablet, with some mice being injected i.p. with PBS, the solvent for MMC, and others being injected i.p. with corn oil, the solvent for DMBA. SCE data obtained on 4 of the 16 control mice were significantly hyperdispersed while a disper-
sion analysis of the pooled control group data indicated that the SCE distribution was not inconsistent with a Poisson model. This was also true when the SCE data among mice implanted with BrdUrd tablets at ORAU and at BNL were combined (Table 9). In spite of the lack of a significant difference in mean SCE frequency between mice exposed to BrdUrd by these two methods, the dispersion of SCE was significantly elevated in tablet bearing mice over BrdUrd-infused mice ( P = 0.02). The biological basis for this increased dispersion in mice implanted with a BrdUrd tablet partially coated with paraffin over mice infused with BrdUrd probably lies in the differences in the time-dependent bioavailability of BrdUrd for incorporation into replicating DNA. The infusion system infuses BrdUrd at a constant rate into the peripheral blood of the recipient mouse (Schneider et al., 1978); thus, the level of BrdUrd in the circulatory system remains relatively constant across time beginning within - 3 0 min of the infusion period (Turturro et al., 1984). In animals implanted with an uncoated BrdUrd tablet, peak circulating BrdUrd levels are obtained - 4 h after tablet implantation, which then decreases over the next 5 h to essentially zero concentrations (King et al., 1982). Coating the tablet with agar (King et al., 1982) or partially coating the tablet with paraffin (McFee et al., 1983) lowers and broadens the peak availability of BrdUrd and incidentally lowers the frequency of SCE. However, even under these conditions, the circulating levels of BrdUrd fluctuate with time. Since SCE are induced by BrdUrd, either directly via incorporation or indirectly by affecting nucleotide pools (Schvartzman and Tice, 1982), it is likely that the increased hyperdispersion of SCE data in mice implanted with a BrdUrd tablet arises from the increased variability in circulating BrdUrd levels among the bone marrow cells. It would be expected that the multiple-injection design of Conner et al. (1978) would have more nearly mimicked the continuous infusion system than the BrdUrd tablet method. The intercellular distribution of SCE induced in vitro or in vivo by many genotoxic agents is hyperdispersed and inconsistent with a Poisson model (Rainaldi and Marriani, 1982; DuFrain, 1983; Whorton et al., 1984; Margolin et al., 1986).
35 TABLE 9 ANALYSIS OF SCE D A T A IN C O N T R O L M I C E Lab.
Method BrdUrd Admin.
Solvent
Number of mice
SCE frequency a Mean + SEM
H b Mean ± SEM
Number of exceptions c
Sum X2 d
df e
Critical X2 f
BNL
IV Infus.
PBS
28
4.76 + 0.217
0.97 _+0.065
2
655.1
672
733.1
BNL
SC Tablet
PBS
4
4.655:0.070
1.39+0.131
1
133.4 *
96
119.9
ORAU
SC Tablet
CO/PBS
16
5.015:0.193
1.27 + 0.116
4
362.5
384
430.4
BNL/ORAU
SC Tablet
CO/PBS
20
4.94-1-0.157
1.29+0.096
5
487.2
480
531.8
Abbreviations: BNL = Brookhaven National Laboratory; O R A U = Oak Ridge Associated Universities; I V = intravenous; SC = subcutaneous; CO = corn oil; PBS = phosphate-buffered saline. a Group mean S C E / c e l l frequency + the standard error of the mean among mice. b Group mean H value + the standard error of the mean among mice. c Numb er of animals where SCE data were significantly different from Poisson expectations. d Sum of Chi square values. e Degrees of freedom. f Critical Chi square value for a = 0.05. * Significantly different at a = 0.05.
While the biological basis for this increasing hyperdispersion with increasing mean SCE frequency has not been resolved, cell-to-cell differences in induced damage, proliferative capacity, cell stage sensitivity, DNA-repair activity a n d / o r available time for repair could modulate the distribution of SCE observed among exposed cell populations (Wolff et al., 1974; Sasaki, 1982; Sharer, 1982; Schvartzrnan and Gutierrez, 1980). For the in vivo study evaluated here, it was considered possible that chemical-specific differences in the pharmacokinetic distribution of reactive species among cycling cells in bone marrow could result in differences in SCE distribution which would not be readily apparent from an analysis of induced SCE frequencies. The 3 carcinogenic chemicals - - CP, DMBA, and MMC - - used in this study were selected for the evaluation of treatment time effects on SCE induction because of their wellestablished genotoxic activity (Preston et al., 1983; Latt et al., 1981) and their differing solubility and metabolic characteristics. The treatment times used were based on the two major approaches to treatment time selection used for in vivo SCE studies. The difference in approach is whether the time of chemical injection occurs around the onset of BrUrd exposure (e.g., Schneider et al., 1978), or around the beginning of the second cell cycle (e.g.,
Allen et al., 1977). The purpose of the former protocol was primarily to increase the opportunity for persistent D N A lesions to induce SCEs in more than one cycle (Tice and Schvartzman, 1982) and to provide time for reactive metabolites to be formed from slowly metabolized chemicals. The purpose of the latter protocol was to avoid the possibility that toxicity could remove heavily damaged cells from the proliferative pool before they had an opportunity to complete the necessary two S phases needed for SCE evaluation (Allen et al., 1977). Treatment of mice with CP and MMC (BNL) induced a significant increase in the dispersion of SCE in bone marrow cells in a dose-dependent manner. The increase in dispersion was also independent of treatment time. Treatment of mice with MMC at ORAU also increased the dispersion in SCE, but the increase was marginally nonsignificant. Comparative analysis by two-way ANOVA using common doses indicated no significant difference between the two laboratories in regard to the dispersion of SCE induced by MMC ( P = 0.78). However, since this analysis includes ~ data only at common doses, data at which the greatest increases in dispersion occurred were not evaluated ( > 2.5 m g / k g MMC). Because the distribution of individual animal SCE data at BNL
36 was different between M M C - t r e a t e d a n i m a l s infused with B r d U r d and those i m p l a n t e d with a B r d U r d tablet, an a d d i t i o n a l t w o - w a y A N O V A was c o n d u c t e d to c o m p a r e the + 1 h tablet d a t a from B N L with + 1 h tablet d a t a of O R A U . The dispersion d a t a were n o t significantly different between the two l a b o r a t o r i e s ( P = 0.12). The lack of a t r e a t m e n t - t i m e effect on the d i s p e r s i o n of S C E i n d u c e d by C P a n d M M C ( B N L ) suggests that there were no significant differences in the p h a r m a c o k i n e t i c d i s t r i b u t i o n of C P or M M C a m o n g different t r e a t m e n t times, no t r e a t m e n t time d e p e n d e n t v a r i a t i o n in cell-stage sensitivity, a n d no cellular toxicity to m o d u l a t e the response. T r e a t m e n t of mice with D M B A also h a d a significant effect on the intercellular d i s t r i b u t i o n of SCE within i n d i v i d u a l animals. However, as o p p o s e d to the o t h e r two chemicals, the increased / - / r e s p o n s e d e p e n d e d on t r e a t m e n t time, with the m a x i m u m H response occurring at the - 1 h t r e a t m e n t time a n d the m i n i m u m response occurring at the + 8 h t r e a t m e n t time. W h i l e the treatm e n t time effects could indicate d i s t r i b u t i o n a l differences in the levels of genotoxic m e t a b o l i t e s present in b o n e marrow, the differences in dispersion could also m e r e l y reflect the m a g n i t u d e of the i n d u c e d SCE yields (see Fig. 2). The d i s p e r s i o n of SCE i n d u c e d b y D M B A differed in o t h e r respects from the d i s p e r s i o n of SCE i n d u c e d b y C P a n d M M C . CP and M M C i n d u c e d a significant increase in H in a d o s e - d e p e n d e n t m a n n e r while the increase in d i s p e r s i o n following the t r e a t m e n t of mice with D M B A was n o t so strictly d e p e n d e n t on dose. Finally, at similar levels of SCE induction, the d i s p e r s i o n in mice treated with D M B A was m u c h greater than that o b s e r v e d in b o n e m a r r o w from mice treated with M M C , a n d to a lesser extent, CP. This difference in d i s p e r s i o n between a w a t e r - i n s o l u b l e c o m p o u n d a n d two water-soluble chemicals is p r o b a b l y d u e to p h a r m a c o k i n e t i c differences in m e t a b o l i s m a n d distrib u t i o n b a s e d on solubility characteristics. It is conceivable that some chemicals, due either to very limited d i s t r i b u t i o n or to highly specific cell stage specificity, m a y i n d u c e a significant increase in dispersion in the absence of a significant increase in m e a n S C E frequency. This analysis suggests that e x a m i n i n g the d i s t r i b u t i o n of SCE, in a d d i t i o n to m e a n frequency, is a useful m e t h o d for
e v a l u a t i n g agent specific p a t t e r n s in SCE induction.
Acknowledgements The a u t h o r s gratefully a c k n o w l e d g e the technical assistance of C. Luke, V. Miller, K. Lowe a n d A. W i l t o n a n d the p r e p a r a t i o n of the m a n u s c r i p t b y M. Rao. This research was s u p p o r t e d b y the National Toxicology Program under NIEHS lnteragency A g r e e m e n t s Y01-ES-20098 ( B N L ) a n d Y01-ES-20100 ( O R A U ) a n d was c o n d u c t e d in l a b o r a t o r i e s which are s u p p o r t e d b y the U.S. Dep a r t m e n t o f E n e r g y ; a c c o r d i n g l y , the U.S. G o v e r n m e n t retains a nonexclusive, royalty-free license to p u b l i s h o r r e p r o d u c e the p u b l i s h e d form of this c o n t r i b u t i o n , or allow others to do so for U.S. G o v e r n m e n t purposes.
References Allen, J.W., C.F. Shuler and S.A. Latt (1978) Bromodeoxyuridine tablet methodology for in vivo studies of DNA synthesis, Somat. Cell Genet., 4, 393-405. Conner, M.K., S.S. Boggs and J.H: Turner (1978) Comparison of in vivo BrdUrd labeling methods and spontaneous sister chromatid exchange frequencies in regenerating murine liver and bone marrow cells, Chromosoma, 68, 303-311. Conner, M.K., Y. Alarie and R.L. Dombroske (1979) Sister chromatid exchange in murine alveolar macrophages, regenerating liver, and bone marrow cells: a simultaneous multicellular assay, Chromosoma, 74, 51-55. Crossen, P.E., J,M. Godwin and M.P. Bodget (1986) Sister chromatid exchange in immature haemopoietic cells, T- and B-lymphocytes, Hum. Genet., 72, 101-103. DuFrain, R.J. (1983) Sister chromatid exchange distribution in rabbit lymphocytes treated with streptonigrin, Environ. Mutagen., 5, 813-824. King, M.-T., D. Wild, E. Gocke and K. Eckhardt (1982) 5-Bromodeoxyuridine tablets with improved depot effect for analysis of sister-chromatid exchanges in bone marrow and spermatogoneal cells, Mutation Res., 97, 117-129. Latt, S.A., J. Allen, S.E. Bloom, A. Carrano, E. Falk, D. Kram, E. Schneider, R. Schreck, R. Tice, B.L. Whitfield, and S. Wolff (1981) Sister chromatid exchanges: A report of the Gene-Tox Program, Mutation Res., 87, 17-62. Littlefield, L.G. (1982) Effects of DNA-damaging agents on SCE, in: A.A. Sandberg (Ed.), Sister Chromatid Exchange, Liss, New York, pp. 355-394. Margolin, B.H., and M.D. Shelby (1985) Sister chromatid exchanges: A reexamination of the evidence for sex and race differences in humans, Environ. Mutagen., 7 (Suppl. 4), 63-72. Margolin, B.H., M.A. Resnick, J.Y. Rimpo, P. Archer, S.M.
37 Galloway, A.D. Bloom and E. Zeiger (1986) Statistical analysis for in vitro cytogenetics assays using Chinese hamster ovary cell, Environ. Mutagen., 8, 183-204. McFee, A.F., K.W. Lowe and J.R. San Sebastian (1983) Improved sister chromatid differentiation using paraffincoated bromodeoxyuridine tablets in mice, Mutation Res., 119, 83-88. Moore II, D.H., and A.V. Carrano (1984) Statistical analysis of high SCE frequency cells in human lymphocytes, in: R.R. Tice and A. Hollaender (Eds.), Sister Chromatid Exchanges, Vol. A, The Nature of SCE, Plenum, New York, pp. 469-479. Perry, P., and H.J. Evans (1975) Cytological detection of mutagen-carcinogen exposure by sister chromatid exchange, Nature (London), 258, 121-124. Rainaldi, R., and T. Mariani (1982) The distribution of induced sister chromatid exchanges: A tool for identifying agents directly interacting with DNA, Mutation Res., 103, 333-337. Santesson, B., K. Lindahl-Kiessling and A. Mattsson (1979) SCE in B and T lymphocytes, Possible implications for Bloom's syndrome, Clin. Genet., 16, 133-135. Sasaki, M. (1982) Sister chromatid exchanges as a reflection of cellular DNA repair, in: A.A. Sandberg (Ed.), Sister Chromatid Exchange, Liss, New York, pp. 35-164. Schneider, E.L., R.R. Tice and D. Kram (1978) Bromodeoxyuridine differential chromatid staining technique: A new approach to examining sister chromatid exchange and cell replication kinetics, in: D.M. Prescott (Ed.), Methods in Cell Biology, Vol. XX, Academic Press, New York, pp. 379-409. Schvartzman, J.B., and C. Gutierrez (1980) The relationship between the cell time available for repair and the effectiveness of damaging treatment in provoking the formation of sister chromatid exchanges, Mutation Res., 72, 483-489. Schvartzman, J.B., and R.R. Tice (1982) 5-Bromodeoxyuridine and its role in the production of sister chromatid exchange, in: A.A. Sandberg (Ed.), Sister Chromatid Exchange, Liss, New York, pp. 123-134. Shafer, D.A. (1982) Alternative replication bypass mechanisms for sister chromatid exchange formation, in: A.A. Sandberg (Ed.), Sister Chromatid Exchange, Liss, New York, pp. 67-98. Sinha, A.K., J.A. Linscombe, B. Gallopude, G.C. Jersey and C.N. Park (1985) Analysis of sister chromatid exchanges in lymphocytes cultured from 71 healthy men, Cell Biol. Toxicol., 1, 333-342.
Snedecor, G.W., and W.G. Cochran (1967) Statistical Methods, 6th edn., Iowa State Press, Ames, IA. Stetka, D.G., J. Minkler and A.V. Carrano (1978) Induction of long-lived chromosome damage, as manifested by sister chromatid exchange in lymphocytes of animals exposed to mitomycin C, Mutation Res., 91, 383-396. Takehisa, S. (1982) Induction of sister chromatid exchanges by chemical agents, in: S. Wolff (Ed.), Sister Chromatid Exchange, Wiley, New York, pp. 87-148. Tice, R.R., and J.B. Schvartzman (1982) Sister chromatid exchange: A measure of DNA lesion persistence, in: A.A. Sandberg (Ed.), Sister Chromatid Exchange, Liss, New York, pp. 33-46. Tice, R.R., T.F. Vogt and D.L. Costa (1982) Cytogenetic manifestations of benzene-induced genotoxic damage in murine bone marrow, in: R.R. Tice, D.L. Costa and K.M. Schaich (Eds.) Genotoxic Effects Of Airborne Agents, Plenum, New York, pp. 257-275. Tice, R.R., A.F. McFee and J.L. Ivett (1987) The effect of agent treatment time on the induction of sister chromatid exchanges in mouse bone marrow cells in vivo, Mutation Res., 182, 15-29. Tice, R.R., J.L. Ivett and A.F. McFee (1989) The effect of agent and agent treatment time on the inhibition of bone marrow cellular proliferation kinetics in vivo, in preparation. Turturro, A , N.P. Singh, J. Bazare Jr. and R.W. Hart (1984) Levels of 5-bromo-2'-deoxyuridine and its metabolites during continuous infusion paradigms in a transplacental system, J. Am. Coll. Toxicol., 3, 73-79. Whorton, E.B., R.R. Tice and D.G. Stetka (1984) Statistical design, analysis and inference issues in studies using SCE, in: R.R. Tice and A. Hollaender (Eds.), Sister Chromatid Exchanges, Vol. A, The Nature of SCE, Plenum, New York, pp. 431-440. Wolff, S., J. Bodycote and R.B. Painter (1974) Sister chromatid exchanges induced in Chinese hamster cells by UV irradiation at different stages of the cell cycle: The necessity of cells to pass through S, Mutation Res., 25, 73-81. Wulf, H.C., B. Husum, H. Engberg-Pedersen and E. Nieburh (1984) Guidelines for the statistical evaluation of SCE, in: R.R. Tice and A. Hollaender (Eds.), Sister Chromatid Exchanges, Vol. A, The Nature of SCE, Plenum, New York, pp. 441-455. Yakovenko, K.N., and V.I. Platonova (1979) Spontaneous level of sister chromatid exchanges and their distribution in human cells, Sov. Genet., 15, 746-753.
25
MUT 04800
The effect of agent dose and treatment time on the intercellular distribution of sister-chromatid exchanges induced by genotoxic agents in mouse bone marrow cells in vivo Raymond R. Tice a Brian G. Ormiston a,. and Alfred F. McFee b a Medical Department, Brookhaven National Laboratory, Upton, N Y 11973 and 6 Medical Sciences Division, Oak Ridge Associated Universities, P.O. Box 117, Oak Ridge, TN 37831 (U.S.A.)
(Received 13 April 1989) (Revision received 7 June 1989) (Accepted 13 June 1989)
Keywords: Sister-chromatid exchanges; Dispersion analysis; Mouse bone marrow; Mitomycin C; Cyclophosphamide; Dimethylbenzanthracene
Summary Using two methods of bromodeoxyuridine (BrdUrd) administration and three genotoxic chemicals, the effects of dose and treatment time on the intercellular distribution of sister-chromatid exchanges (SCE) in the bone marrow of male B6C3F1 mice were evaluated. The dispersion of SCE among solvent control mice infused intravenously with BrdUrd or implanted subcutaneously with a BrdUrd tablet partially coated with paraffin was largely consistent with a Poisson model. Intraperitoneal treatment with cyclophosphamide (CP; solvent = phosphate-buffered saline), 7,12-dimethylbenzanthracene (DMBA; solvent = corn oil) and, in mice infused with BrdUrd, mitomycin C (MMC; solvent = phosphate-buffered saline) induced a significant increase in SCE, the distribution of which was not distributed as a Poisson. For CP and MMC, the increase in dispersion was dose-dependent and independent of treatment time ( - 1 , + 1 or + 8 h in relation to the start of the BrdUrd treatment). The lack of a treatment time effect suggests that there were no significant differences among treatment times in the distribution of the reactive forms of these two chemicals, no variation in cell-stage sensitivity, and no cellular toxicity to modulate the response. For DMBA, the increased dispersion of induced SCE depended on treatment time and was not simply related to dose. The increase in dispersion was agent-specific; at equal levels of SCE induction, the distribution of SCE in mice treated with DMBA exhibited greater dispersion than SCE in mice treated with either CP or MMC. These differences between DMBA and C P / M M C are probably due to DMBA's slower absorption/distribut!on kinetics, its requirement for metabolic activation to genotoxic metabolites and its extended half-life. These data suggest that analyzing the distribution of SCE, in addition to mean frequency, is a useful method for evaluating agent specific patterns in SCE induction.
Present address: Environmental Services Division, Reynolds, Smith and Hills, Inc., P.O. Box 22003, Tampa, FL 33622
(U.S.A.).
Correspondence: Dr. Raymond Tice, Integrated Laboratory Systems, P.O. Box 13501, Research Triangle Park, NC 27709 (U.S.A.).
0027-5107/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
26 The observed frequency of sister-chromatid exchanges (SCE) by a genotoxic chemical depends on the molecular target of the inducing agent (Perry and Evans, 1975; Littlefield, 1982; Takehisa, 1982), the extent of lesion persistence (Stetka et al., 1978; Sasaki et al., 1982; Shafer, 1982; Tice and Schvartzman, 1982), the temporal relationship between the formation of the DNA damage and the onset of scheduled DNA synthesis (Stetka et al., 1978; Schvartzman and Gutierrez, 1980; Sasaki, 1982; Shafer, 1982), and the extent of cell lethality associated with the treatment. The observed frequency of SCE in vivo also depends on various pharmacokinetic considerations (e.g., route of exposure, reactive species half-life) and intrinsic factors such as species, strain, sex, age of the animal (Tice et al., 1982), and the target tissue (Conner et al., 1979). Recently, we investigated the effect of treatment time in relation to the onset of BrdUrd exposure on the magnitude of the SCE response generated by treating mice with one of three well-known genotoxic chemicals - c y c l o p h o s p h a m i d e (CP), 7,12-dimethylbenzanthracene (DMBA) and mitomycin C (MMC) (Tice et al., 1987). In that study, the effect of the method of bromodeoxyuridine (BrdUrd) administration (i.e., intravenous infusion versus a paraffin-coated tablet implanted subcutaneously) on the induction of SCE by MMC, as well as interlaboratory differences in response, were examined. We found that the induction of SCE, as measured by individual animal mean SCE frequency, was independent of the method of BrdUrd administration and independent of (for MMC) or dependent on (for CP and DMBA) treatment time (Tice et al., 1987). In 1982, Rainaldi and Mariani published a paper in which they discussed the importance of evaluating the intercellular dispersion of in vitro SCE data as a method for discriminating among agents with differing mechanisms of action. More recently, Margolin and Shelby (1985) demonstrated that groups of individuals with nonsignificantly different SCE means could have significantly different SCE/cell dispersions. These studies prompted us to re-evaluate the SCE data obtained in the in vivo mouse bone marrow treatment time study for the effect of chemical agent, treatment time and method of BrdUrd administration on the intercellular distribution of SCE within
individual animals. We anticipated that this analysis would provide information on the distribution of SCE among cells in control mice, as well as insight into the intercellular distribution of DNA damage induced by three different genotoxic chemicals. Materials and methods
A complete description of the materials and methods used in this study can be found in Tice et al. (1987). Male B6C3F1 mice (Frederick Cancer Research Center, Frederick, MD), between 10 and 14 weeks of age, were used. Food (Purina Laboratory Chow 5001) and water were provided ad libitum. Mitomycin C (CAS No. 50-07-7), BrdUrd, and reagent-grade corn oil were purchased from Sigma; colchicine from Eli Lilly, CP (CAS No. 50-18-0; Cytoxan) from Mead Johnson, DMBA (CAS No. 57-97-6) from Eastman Kodak, Hoechst 33258 from American Hoechst, Giemsa from Harleco. MMC and CP were dissolved in phosphate-buffered saline (PBS; pH 7.4); DMBA was suspended in corn oil. All injections were intraperitoneal (i.p.) at a volume of 0.1 ml (MMC) or 0.2 ml (CP, DMBA). In some experiments conducted at Brookhaven National Laboratory (BNL), individually restrained mice were continuously infused intravenously (i.v.) for 24 h with BrdUrd (50 m g / k g / h ) in PBS, while in other experiments at BNL and at Oak Ridge Associated Universities (ORAU), a 50 mg BrdUrd tablet, partially coated (70%) with paraffin (McFee et al., 1983) was implanted subcutaneously (s.c.). At 1 h before, 1 h after, or 8 h after the beginning of exposure to BrdUrd, groups of animals were injected i.p. with one of the three chemicals or the appropriate solvent. 20-22 h after the beginning of BrdUrd exposure, each animal was injected with colchicine. 2 h later, the mice were killed by cervical dislocation, femoral bone marrow was removed, exposed to hypotonic solution and fixed using 3 : 1 methanol : glacial acetic acid. Coded slides were prepared, differentially stained and 25 second-generation metaphase cells per animal were analyzed for SCE.
Statistical analysis. For all statistical analyses, significance was determined at an alpha level of
27 0.05. T o c o m p a r e the effects of dose and treatm e n t time o n the intercellular d i s t r i b u t i o n of S C E within individual animals, the dispersion coefficient H ( S n e d e c o r and C o c h r a n , 1967) w h i c h is the ratio of the s a m p l e variance to the s a m p l e m e a n ( M a r g o l i n an d Shelby, 1985) was analyzed by two - way A N O V A . T o d e t e r m i n e w h e t h e r the distribution of S C E within individual animals fit a Poisson model, the dispersion test ( S n e d e c o r a n d C o c h r a n , 1967) was applied. I n this a p p r o a c h , the statistic T = ( r - 1 ) H , w h e r e r is the n u m b e r of cells analyzed for SCEs, was calculated. T h e statistic T is referred to a table of critical values for the C h i - s q u a r e d i s t r i b u t i o n with r - 1 degrees of f r e e d o m ( M a r g o l i n et al., 1986). T o d e t e r m i n e w he the r the S C E f r e q u e n c y d i s t r i b u t i o n for groups of mice used as controls or treated with various doses of the car ci no g e n s fit a Poisson model, animals treated with the same dose were e x a m i n e d collectively by the dispersion test, c o m b i n i n g where a p p r o p r i a t e across t r e a t m e n t time groups. In addition, the co r r el at i o n b e t w e e n H and S C E
f r e q u e n c y was d e t e r m i n e d using linear regression analysis, c o m b i n i n g d a t a across t r e a t m e n t times w h er e a p p r o p r i a t e .
Results
Cyclophosphamide (BNL). T h e S C E d a t a for each animal, by dose an d t r e a t m e n t time, along with the i n d i v i d u a l m o u s e m e a n S C E f r e q u e n c y a n d the c a l c u l a t e d dispersion coefficient H are p r o v i d e d in T a b l e 1. C P at doses of 6.25, 12.5 an d 25.0 m g / k g i n d u c e d a linear increase in S C E f r e q u e n c y in mice i n f u sed with B r d U r d (Tice et al., 1987). T h e slope of the S C E response d e p e n d e d on t r e a t m e n t time. W h e n the f r e q u e n c y distribution of S C E d a t a within each c o n t r o l a n i m a l was analyzed b y the dispersion test for goodness of fit with a Poisson model, only 1 of 11 an i m al s differed significantly f r o m e x p e c t a t i o n s ( T a b l e 1). T r e a t m e n t with C P i n d u c e d a significant increase in the dispersion of S C E within i n d i v i d u a l animals ( P < 0.01) w h i ch was m a r g i n a l l y i n d e p e n d e n t of
TABLE 1 INDUCTION OF SCE BY CYCLOPHOSPHAMIDE Dose
-1 h a
(mg/kg)
Mean +_SEM b
0
+1 h a Range c
Hd
+8h a
Mean -+SEM b
Range c
Hd
Mean -+SEM b
Range c
Hd
1- 7 0- 8 2- 9 1- 14
0.75 1.18 0.82 1.49
4.00+0.38 4.00+_0.42 6.04+0.69 6.12+0.52
1- 7 1- 9 2-18 1-11
0.90 1.11 1.95 * 1.10
2.80-+0.28 3.96+0.39 7.20+0.42
0- 6 2- 8 4-11
0.71 0.94 0.63
12.48+0.64 16.44+1.00 16.80+_1.28 17.16_+0.86
7-19 10-31 6-33 9-24
0.82 1.53 * 2.44 * 1.07
13.40-+0.85 13.92-+0.80 16.12-+0.93 19.24_+1.32
6-27 8-22 7-26 7-36
1.34 1.16 1.35 2.26 *
22.20-+ 1.40 25.00-+1.26 25.36-+1.99 26.16_+1.33
815816-
34 40 47 40
2.20 1.58 3.90 1.69
12.5
21.08+0.98 26.44+1.10 29.76+_1.50 31.64+_2.22
12-30 15-38 17-49 4-51
1.13 1.13 1.89 * 3.90 *
24.56-+1.65 28.96+2.27 29.88+1.66 38.20+1.95
12-44 13-62 20-51 21-59
2.78 4.44 2.30 2.48
* * * *
36.24+1.88 36.68 + 3.01 39.68+2.03 47.00-+1.67
2291933-
58 79 64 64
2.45 * 5.86 * 2.60 * 1.49
25.0
41.16+-3.90 46.16 +-2.20 49.32+_2.80 59.52+3.19
13-85 29-70 22-73 32-89
9.25 2.63 3.96 4.27
50.00+-2.76 50.60 +-3.09 51.96+_1.66 65.28+_2.43
28-84 23-81 35-69 44-93
3.80 * 4.72 * 1.33 2.25 *
55.80 + 2.49 61.00+2.89 61.20+3.58 75.44+2.84
37- 84 31- 84 28- 92 48-109
6.25
* * * *
a Treatment time in relation to the onset of exposure to BrdUrd. b Animal mean SCE/cell frequency 5: standard error of the mean among 25 cells. Range of SCE values. d Dispersion coefficient = variance/mean. * Significantly different at a = 0.05 from Poisson distribution.
2.96+0.30 4.08_+0.44 5.04+0.41 7.08+0.65
2.77 3.43 5.23 2.67
* * * *
* * * *
28 TABLE 2 DI SPERSION ANALYSIS OF C P - I N D U C E D SCE DATA
/ /
Dose (mg/kg)
Nu mb er of animals
Number ofexceptions ~
Sum b X2
df ~
/
Critical J X2
0
11
1
277.8
264
302.6
6.25
12
7
646.6 *
288
328.3
12.50
12
9
778.7 •
288
328.3
25.0
12
11
1119.9 •
288
328.3
//I / •
treatment time ( P = 0.06). A dispersion analysis of SCE data from mice at each dose combined across treatment times indicated that while the 'I
•
l--
I.
•
•
II
AOA
.
•
/
J
i .I ~-
Data combined for treatment times - 1 , + 1, + 8 h. Number of animals whose SCE data were significantly different from Poisson expectations. h Sum of Chi square values. ~ Degrees of freedom. d Critical Chi square value for a = 0.05. * Significantly different at a = 0.05.
tD
/
•
i A
/Oo- : i
o
SCE FREQUENCY
Fig. 2. Plot of the correlation between SCE frequency and the dispersion correlation H for dimethylbenzanthracene. Each data point represents the H value and SCE frequency for an individual mouse basedon an analysisof 25 second-generation metaphase cells. Square ~ - l h treatment time data. Linear regression analysis of the data indicated a significant regression of H against SCE frequency (regression coefficient r = 0.6059; P = 0.0167; y ~ 0.917 +0.096 x denoted by the solid line); Circle = + 1 h treatment time data. Linear regression analysis of the data indicated a significant regression of H against SCE frequency (regression coefficient r = 0.7207; P = 0.0007; y = 0.415+0.084 x denoted by the dashed line); Triangle = + 8 h treatment time data. Linear regression analysis of the data indicated a significant regression of H against SCE frequency (regression coefficient r = 0.7859; P = 0.0003; y = 0.005 +0.169 x denoted by the dash-dotted line). Solid symbols indicate SCE distributions for an individual mouse significantly different from Poisson expectations at c~= 0.05.
8o
data for control animals fit a Poisson model, the distribution in CP-treated mice differed significantly from Poisson expectations at each dose (Table 2). With increasing dose, the SCE response became increasingly hyperdispersed (Fig. 1).
Fig. 1. Plot of the correlation between SCE frequency and the dispersion correlation H for cyclophosphamide. Each data point represents the H value and SCE frequency for an individual mouse based on an analysis of 25 second-generation metaphase cells. Square = - 1 h treatment time data; Circle = + 1 h treatment time data. Triangle = + 8 h treatment time data. Linear regression analysis of the data pooled across treatment times indicated a significant regression of H against SCE frequency (regression coefficient r = 0.5724; P < 0.0001; y =1. 015+0.0 48 x denoted by the solid line). Solid symbols indicate SCE distributions for an individual mouse significantly different from Poisson expectations at a = 0.05.
Dimethylbenzanthracene (ORAU). The individual cell SCE data, along with each animal's mean frequency and the calculated H statistic, for mice treated with DMBA at doses of 0, 10, 50 and 300 m g / k g at - 1 , + 1 and + 8 h relative to the time of BrdUrd tablet implantation can be found in Table 3. The shape and magnitude of the SCE dose-response for DMBA was highly dependent upon treatment time (Tice et al., 1987). With decreasing time between treatment and sampling
i•
o
L
,o
•
2'0
3'o
4'0
•
•
i
5o
•
m
6o
;o
8CE FREQUENCY
29 TABLE 3 INDUCTION
OF SCE BY 7,12-DIMETHYLBENZANTHRACENE
Dose
-1 h a
(mg/kg)
Mean ± SEM b
Range ~
H d
M e a n -+ S E M b
0
3.96+_0.40 4.00-+0.49 6.72_+0.85
0-11 1- 8 0-16
1.53 * 0.98 2.71 *
10
11.60_+0.85 13.72_+1.24 14.92_+1.17 15.08_+1.43
4-20 5-30 7-27 5-31
1.55 2.80 2.28 3.40
50
15.20+_1.02 15.44_+1.07 17.56_+1.10 19.96 + 1.08
100
26.16_+1.96 28.24_+1.96 29.64_+1.42 29.68 _+ 2.66
a b c d *
+8h
+1 h a Range c
H a
a
Mean + SEM b
Range c
H d
4.56 ± 0.42 4.84+0.51 4.92+0.56 5.16+0.44
1- 9 1-10 1-11 1- 9
0.95 1.34 1.61 * 0.93
4.28_+0.41 4.52_+0.46 4.60_+0.45 4.76_+ 0.39 5.12_+0.44
1- 9 0-10 1-11 0- 8 1-11
0.98 1.18 1.12 0.81 0.95
* * * *
11.76_+0.65 11.92_+0.79 13.96_+0.82 15.40+_1.16 15.48 -+ 0.94
7-20 5-21 7-25 7-30 8-27
0.91 1.32 1.19 2.19 • 1.42
10.16+_0.89 12.04+0.77 12.08 + 1.10 23.04+2.09
2-21 6-19 3-23 9-47
1.95 * 1.24 2.52 * 4.76 *
8-26 7-29 8-30 11-33
1.73 * 1.86 * 1.72 * 1.46
16.36-+0.84 17.20_+1.25 20.32_+1.27 21.68 _+ 1.45
9-27 9-31 10-34 8-36
1.07 2.28 • 1.98 • 2.42 •
10.88_+0.83 11.96+0.81 12.56_+0.79 13.24_+0.75
5-21 6-23 5-21 7-24
1.56 * 1.37 1.24 1.06
11-48 13-73 20-51 16-75
3.68 4.54 1.69 5.95
20.52_+0.82 22.32_+1.45 22.60_+1.37 25.96 + 2.06
12-29 9-36 11-39 14-56
0.82 2.37 • 2.09 • 4.10 •
10.44_+0.87 11.44_+0.91 12.56_+1.01 16.12_+1.51
3-19 4-20 4-23 8-32
1.83 1.80 2.04 3.53
* * * *
* * * *
T r e a t m e n t time in relation to the onset of exposure to BrdUrd. A n i m a l m e a n S C E / c e l l frequency +_ standard error of the m e a n a m o n g 25 cells. R a n g e of SCE values. D i s p e r s i o n coefficient = v a r i a n c e / m e a n . Significantly different at a = 0.05 from Poisson distribution.
times, the magnitude of the SCE response was diminished and the shape of the dose response increasingly curvilinear. Both D M B A dose ( P < 0.01) and treatment time ( P = 0.04) had a significant effect on the dispersion of SCE within individual animals. Pairwise comparisons among the 3 treatment times indicated that the injection of mice with D M B A 1 h prior to BrdUrd tablet implantation induced a greater dispersion in SCE than any other treatment time. The presence of a treatment time effect prevented the combining of data within doses across treatment times. A dispersion analysis of the individual dose by treatment time group data indicated that only the data for the + 1 h treatment time control group was Poisson distributed (Table 4). When H was regressed against SCE frequency for each animal treated with D M B A within each treatment time (Fig. 2), a complex relationship between the in-
duced SCE response and the SCE distribution became apparent.
Mitomycin C (ORAU). The SCE data for animals implanted with a BrdUrd tablet and treated with M M C are given in Table 5. In addition to treatment times of - 1 , + 1 and + 8 h in relation to BrdUrd tablet implantation, some mice were injected i.p. with M M C at the time of BrdUrd treatment (i.e., at time 0). The mean SCE response for M M C at all treatment times was linear over the dose range tested (1.0-4.0 m g / k g ) , and without a significant treatment time effect (Tice et al., 1987). Analysis of the individual mouse SCE distribution data for goodness of fit to a Poisson model revealed that, with increasing dose, an increasing number of animals yielded data which deviated significantly from Poisson expectations (Table 5). Analysis by two-way A N O V A indicated
30
that neither the treatment time ( P -- 0.49) nor the treatment ( P = 0.08) had a significant effect on the dispersion of SCE data within individual mice. Therefore, all animals were combined across treatment time and dose and collectively analyzed by the dispersion test. A significant difference from a Poisson model was obtained (Table 6). A plot of the H statistic against each animal's respective SCE data is presented in Fig. 3.
g •
•
o
Mitomycin C (BNL). The individual cell SCE data by treatment time, method of BrdUrd administration, dose, and animal are provided in Table 7. MMC at doses of 1.0, 2.0, 2.5 and 3.0 m g / k g
~0
6 DISPERSION DATA
OF
ANALYSIS
DMBA-INDUCED
SCE
(A) T r e a t m e n t time = - 1 h
0 10 50 300
~'
o
25
3b
i
4o
i
50
65
SCE FREQUENCY
TABLE 4
Dose (mg/kg)
0
2o
Number of
Number o f excep-
animals
tions
3 4 4 4
2 4 3 4
Sum X2 b
df c
Critical d X2
a
125.1 240.9 162.5 380.7
* * * *
72 96 96 96
92.8 119.9 119.9 119.9
df c
Critical a
Fig. 3. Plot o f the c o r r e l a t i o n b e t w e e n S C E f r e q u e n c y and the dispersion c o r r e l a t i o n H f o r m i t o m y c i n C ( O R A U ) . E a c h d a t a point represents the H v a l u e a n d S C E f r e q u e n c y for a n individual m o u s e b a s e d o n a n a n a l y s i s o f 25 s e c o n d - g e n e r a t i o n m e t a p h a s e cells. S q u a r e = - 1 h t r e a t m e n t t i m e d a t a ; D i a m o n d = 0 t r e a t m e n t t i m e d a t a ; Circle = + 1 h t r e a t m e n t t i m e d a t a ; Triangle = + 8 h treatment time data. Linear regression analysis of the d a t a p o o l e d a c r o s s t r e a t m e n t times did n o t result in a s i g n i f i c a n t r e g r e s s i o n o f H against S C E f r e q u e n c y (regression c o e f f i c i e n t r = 0.2068; P = 0.1039; y = 1 . 1 0 7 + 0 . 0 0 6 x den o t e d by the solid line). Solid s y m b o l s i n d i c a t e S C E distributions for a n i n d i v i d u a l m o u s e s i g n i f i c a n t l y d i f f e r e n t f r o m Poisson e x p e c t a t i o n s at a = 0.05.
(B) T r e a t m e n t time = + 1 h Dose (mg/kg)
0 10 50 300
Number of
Number of excep-
animals
tions a
5 5 4 4
0 1 3 3
Sum X2 b
X2 121.6 168.9 * 186.2 * 225.2 *
120 120 96 96
146.3 146.3 119.9 119.9
Sum X2 b
df ~
Critical d
(C) T r e a t m e n t t i m e = + 8 h Dose (mg/kg)
0 10 50 300
Number of
Number of excep-
animals
tions
4 4 4 4
1 3 1 4
X2
a
115.8 251.3 * 125.8 * 220.9 *
96 96 96 96
119.9 119.9 119.9 119.9
N u m b e r of a n i m a l s w h o s e S C E d a t a were s i g n i f i c a n t l y different f r o m P o i s s o n e x p e c t a t i o n s . b S u m of Chi s q u a r e values. L. D e g r e e s of f r e e d o m . d Critical C h i s q u a r e v a l u e for c~= 0.05. * S i g n i f i c a n t l y d i f f e r e n t at a = 0.05.
induced a linear increase in SCE frequency which was independent of treatment time and of the method of BrdUrd administration (Tice et al., 1987). Analyzing the dispersion of SCE in individual animals resulted in finding a number of animals in which the SCE response failed to agree with a Poisson model (Table 7). Analysis of H by A N O V A using dose and treatment time or dose and method of BrdUrd administration as factors indicated for mice infused with BrdUrd that treatment with M M C resulted in a significant increase in H ( P < 0.01) which was independent of treatment time ( P = 0.11). A dispersion analysis of mouse data pooled across treatment times indicated a significant departure from a Poisson model at every dose of MMC (Table 8A). In control mice implanted with BrdUrd tablets, SCE were not distributed as a Poisson ( P < 0.01) and treatment with M M C marginally affected the dispersion of SCE (Table 8B). A graphic presentation
TABLE 5 I N D U C T I O N OF SCE BY M I T O M Y C I N C (ORAU) Dose
-lh
(mg/kg)
Mean -+ SEM b
a
Oh a Range c
H d
0
Mean + SEM b
+lh Range c
n d
4.96 + 0.47 5.76_+0.61 5.80+_0.48 6.20 ± 0.60
1- 9 2 13 1- 9 1-12
1.12 1.61 * 1.01 1.47
a
+8h a
Mean + SEM b
Range c
H a
Mean +_ SEM b
Range c
H d
1.0
15.80-+0.85 16.04-+0.72 16.32+0.95 17.00-+0.85
1010911-
28 25 29 31
1.14 0.80 1.39 1.07
11.32+0.73 14.52+0.86 18.44-+0.77 19.36-+1.11
7-22 6-24 13-26 10-32
1.18 1.26 0.81 1.58 *
14.28+0.84 16.28-+1.11 17.764-0.85 18.32_+0.95
9-28 6-28 11 27 11-30
1.23 1.90 * 1.02 1.23
15.60-t-0.56 15.88 -+ 0.90 16.00--+0.93 16.08 ± 0.72
11-20 9-27 8-28 9-22
0.50 1.27 1.36 0.80
2.5
16.24-t-0.61 19.60_+0.68 19.68-+0.95 24.80_+0.99
1012918-
21 28 31 37
0.58 0.58 1.14 0.98
15.88-+1.08 23.36+1.12 24.96+0.93 25.72_+1.03
9-29 12-40 17-39 17-36
1.82 * 1.34 0.87 1.04
25.04-+0.95 33.84-+1.23 39.60 + 1.35 41.80-+ 1.83
14-34 23-48 29-61 26-62
0.90 1.12 1.14 2.00 *
23.56_+0.95 24.764-0.99 39.56-+ 1.18 39.60 + 1.29
18-39 17-38 29-53 29-53
0.95 0.99 0.88 1.04
4.0
36.96+1.76 43.24_+1.69 49.92-+1.65 53.00_+3.01
22- 58 3 1 - 60 35- 71 36-110
2.10 * 1.64 * 1.37 4.27 *
41.08_+1.48 42.84_+1.28 49.60_+1.48
26-53 29-58 36-65
1.33 0.96 1.11
44.36-+1.19 50.52 + 1.51 51.04 + 1.76 59.64+ 1.65
30-55 37-65 37-73 48-76
0.80 1.13 1.52 * 1.15
41.32 + 1.35 49.56 + 1.35 50.52 -+ 1.51 54.68-+2.02
29-53 38-67 37-65 36-77
1.09 1.24 1.13 1.86 *
a b c d *
Treatment time in relation to the onset of exposure to BrdUrd. Animal mean S C E / c e l l frequency+ standard error of the mean a m o n g 25 cells. Range of SCE values. Dispersion coefficient = v a r i a n c e / m e a n . Significantly different at a = 0.05 from Poisson distribution.
32 TABLE 6 DISPERSION A N A L Y S I S OF M M C - I N D U C E D SCE DATA
(ORAU) Number of animals
Number of exceptions a
Sum b X2
df c
Critical o X2
52
10
1568.8 *
1248
1331.0
Data combined across doses and across treatment times of - 1 , 0 , +1, + 8 h . " Number of animals whose SCE data were significantly different from Poisson expectations. b Sum of Chi square values. c Degrees of freedom. d Critical Chi square value for a = 0.05. * Significantly different at a = 0.05.
tion of SCE in control mice injected with BrdUrd for 9 consecutive hours was consistent with Poisson expectations. At BNL, the majority of the mice were i.v. infused with BrdUrd dissolved in PBS, a limited number of mice were implanted s.c. with a BrdUrd tablet partially coated with paraffin. All control mice were injected i.p. with PBS, the solvent for MMC and CP. Among the 28 BrdUrd infused, solvent control mice, only 2 mice provided SCE data which were inconsistent with a Poisson model and analyzing the BrdUrd infused control animals by the dispersion test indicated that the pooled SCE data were distributed as a Poisson (Table 9).
of the relationships between H and SCE frequency for MMC can be found in Fig. 4. 4
Discussion
In vitro, the intercellular distribution of SCE within individual control culture dishes of V79 cells (Rainaldi and Mariani, 1982) or of CHO cells (Margolin et al., 1986), both functionally homogeneous populations, can be satisfactorily described by a Poisson model. In human peripheral blood lymphocytes mitogenically stimulated to proliferate in vitro, a Poisson distribution is inadequate for modelling the dispersion of SCE data within some individuals (Yakovenko and Platonova, 1979; Wulf et al., 1984; Whorton et al., 1984; Moore and Carraro, 1984; Sinha et al., 1985; Margolin and Shelby, 1985). This lack of fit suggests heterogeneity in sensitivity to BrdUrd among subpopulations of responding cells (Santesson et al., 1979; Crossen et al., 1986) a n d / o r prior exposure of the donor to genotoxic agents (Moore and Carrano, 1984). The latter explanation may be more likely because in a study involving mitogenically-stimulated rabbit lymphocytes, a dispersion analysis confirmed the adequacy of the Poisson model for describing the distribution of baseline SCE (DuFrain, 1983). Since bone marrow is comprised of several functionally distinct cell populations, it might be anticipated that the intercellular distribution of SCE values would be inconsistent with a simple Poisson model. However, Conner et al. (1978) concluded that the intercellular distribu-
3, ~ ~g~
~V , ,
0
Q
m
A
•
A
.
&
0
q)
,6>
•
z~
0
o
•
G
2'0
.~
4~
~
6~
8CE FREQUENCY
Fig. 4. Plot of the correlation between SCE frequency and the dispersion correlation H for mitomycin C (BNL). Each data point represents the H value and SCE frequency for an individual mouse based on an analysis of 25 second-generation metaphase cells. Square = - 1 h treatment time data for BrdUrd-infused mice; Circle = + 1 h treatment time data for BrdUrd-infused mice; Triangle = + 8 h treatment time for BrdUrd-infused mice. Linear regression analysis of the data pooled across treatment times indicated a significant regression of H against SCE frequency (regression coefficient r = 0.4762; P < 0.0001; y = 0 . 8 6 8 + 0 . 0 2 3 x denoted by the solid line). Diamond = +1 h treatment time data based on BrdUrd tablet-implanted mice. Linear regression analysis of the data pooled across treatment times did not result in a significant regression of H against SCE frequency (regression coefficient r = 0 . 2 8 3 5 ; P = 0 . 2 8 7 2 ; ) , = 1 . 4 2 6 + 0 . 0 1 0 x denoted by the dashed line). Solid symbols indicate SCE distribution for an individual mouse significantly different from Poisson expectations at a = 0.05.
a
18.92-+1.00 19.80_+ 1.36 20.56-1-1.13 22.76+1.80 23.92+1.22
24.80_+1.57 25.56+1.93 25.88 + 0.71 29.40-+ 1.52 35.64 + 0.97
34.48 + 1.63 36.36+1.72 42.28 5:1.34 53.00-+2.07 58.16+2.27
2.0
2.5
3.0
19-49 21- 5 6 31-60 34- 7 2 38-83
2-42 9-54 1 9- 34 13-53 28-45
11-28 9- 3 3 10-32 3-40 13-39
5-21 5-20 6-23 5-22 7-19
1- 7 2- 7 2- 9 1- 8 1-13 2-10 2-12
Range c
* * * *
1.93 2.04 1.06 2.02 2.21 * *
* *
2.48 * 3.64 * 0.49 1.96 * 0.66
1.32 2.33 1.56 3.55 1.55
1.50 1.32 1.15 1.23 1.18
0.67 0.47 0.92 0.93 1.84 * 0.97 0.84
H d
a
37.96 _+1.33 38.44_+1.62 47.92 -+ 2.32 53.36_+2.13
26.40_+1.09 27.84_+1.15 29.92 + 1.33 30.68 -+ 1.38 31.00-+ 0.87
19.52_+1.11 22.48 + 1.43 22.92-+0.86 23.56+1.10 24.92_+1.35
13.64-+0.66 3.84-+0.53 14.04 _+0.61 14.88 5:0.53 16.68-+0.81
3.96-+0.42 4 .08+ 0.29 4.20-+0.29 4.44+0.42 4.56_+0.44
Mean _+SEM b
+lh
9 7 7 9 9
25-52 25-59 30-82 40-82
18-37 13-36 18-47 20-49 23-39
12-32 12-36 14-30 15-36 15-43
8-22 10-20 9-21 10-21 9-24
02111-
Range ¢
1.17 1.72 * 2.81 * 2.12 *
1.13 1.19 1.48 1.56 * 0.61
1.58 * 2.28 * 0.81 1.28 1.83 *
0.80 0.52 0.66 0.48 0.97
1.13 0.51 0.52 1.00 1.06
H a
Treatment time in relation to the onset of exposure to BrdUrd. A ni m al mean S C E / c e l l frequency+ s ta nda rd error of the mean a m o n g 25 cells. Range of SCE values. Dispersion coefficient = variance~mean. Significantly different at a = 0.05 from Poisson distribution.
11.44+0.83 11.72+0.79 12.04 + 0.74 12.12 + 0.77 12.72:t:0.78
1.0
a b c a *
3.76+0.27 3.92-+0.32 4.32+0.40 4.64+0.42 5.445:0.63 6.08 + 0.49 6.72-1-0.47
Mean + SEM b
-lh
BrdUrd infusion
0
Dose (mg/kg)
I N D U C T I O N OF SCE BY M I T O M Y C I N C (BNL)
TABLE 7
40.52 _+ 1.33 41.40 + 1.94 45.56 + 2.25 45.76 -+ 1.34 48.80-+2.35
30.48 + 1.17 32.48 _+1.31 34.12 _+ 1.13 35.40 _+1.41 38.32_+1.39
25.68_+1.39 27.76 + 1.02 28.16 5:1.40 28.40 _+1.54 29.36+0.91
15.16+0.88 18.36_+1.14 18.48 _+ 1.09 19.52 _+0.96 21.40 __.0.91
3.76_+0.42 4.44+ 0.40 4.88 _+0.44 5.04_+0.43 5.64_+0.45
Mean + SEM b
+8h a
26-53 27-64 24-73 32-57 26-84
22-47 21-50 22-43 24-51 22-52
12-37 19-37 18-46 16-46 22-39
6-24 10-32 3-27 9-29 11-30
0- 8 1- 8 0- 9 1- 9 2-12
Range ¢
1.09 2.28 * 2.79 * 0.98 2.84 *
1.13 1.32 0.93 1.41 1.25
1.87 * 0.94 1.73 * 2.09 * 0.71
1.27 1.76 * 1.61 * 1.19 0.97
1.16 0.88 1.00 0.92 0.90
H a
a
33.68_+ 1.84 34.20 _+ 1.41 38.76 5:1.72 40.52 + 1.48
30.28 _+ 1.47 30.44 + 1.41 30.52_+1.43 36.16_+1.58
15.88_+0.85 18.32_+1.32 18.64_+1.31 18.68_+0.92
4.48+0.44 4.60+0.56 4.72+0.50 4.80+0.53
M ean -+ SEM b
+lh
Br d U r d tablet
19-54 18-47 24-55 26-52
15-47 21-43 17-54 26-53
9-23 6-33 6-35 11-28
1- 9 1-13 1-11 1 11
Range c
2.52 * 1.45 1.91 * 1.35
1.78 * 1.41 1.67 * 1.73 *
1.15 2.39 * 2.29 * 1.13
1.10 1.72 * 1.30 1.44
H a
34 TABLE 8 DISPERSION ANALYSIS OF MMC-INDUCED (BNL)
SCE DATA
(A) B r d U r d i.v. i n f u s i o n m e t h o d Dose (mg/kg)
0 1.0 2.0 2.5 3.0
Number of
Number o f excep-
animals
tions a
17 15 15 15 14
1 2 10 3 10
Sum b X2
df c
377.3 503.0 610.3 509.8 649.4
408 360 360 360 336
Critical d X2
* * * *
455.8 405.0 405.0 405.0 379.5
D a t a c o m b i n e d f o r t r e a t m e n t times - 1 , + 1, + 8 h. ~' N u m b e r o f a n i m a l s w h o s e S C E d a t a w e r e s i g n i f i c a n t l y different f r o m P o i s s o n e x p e c t a t i o n s . b S u m of C h i s q u a r e values. " Degrees of freedom. d Critical Chi s q u a r e value f o r a = 0.05. * S i g n i f i c a n t l y d i f f e r e n t at a = 0.05. (B) B r d t J r d t a b l e t m e t h o d Dose (mg/kg)
Number of animals
Number o f exceptions a
Sum b X2
0 1.0 2.5 3.5
4 4 4 4
1 2 3 2
133.4 167.0 158.l 173.5
h c d *
* * * *
Number of animals whose SCE data different from Poisson expectations. S u m of C h i s q u a r e values. D e g r e e s of f r e e d o m . Critical Chi s q u a r e v a l u e for a = 0.05. S i g n i f i c a n t l y d i f f e r e n t at a = 0.05.
df c
Critical d X2
96 96 96 96
119.9 119.9 119.9 119.9
were s i g n i f i c a n t l y
In mice at BNL implanted s.c. with a BrdUrd tablet, the SCE distribution was significantly hyperdispersed in 1 of 4 mice and the pooled group data were inconsistent with Poisson expectations (Table 9). This apparent difference between the intercellular distribution of SCE values within control mice depending on the method of BrdUrd administration occurred in spite of a lack of difference in the mean SCE frequency. At ORAU, all solvent control animals were implanted s.c. with a BrdUrd tablet, with some mice being injected i.p. with PBS, the solvent for MMC, and others being injected i.p. with corn oil, the solvent for DMBA. SCE data obtained on 4 of the 16 control mice were significantly hyperdispersed while a disper-
sion analysis of the pooled control group data indicated that the SCE distribution was not inconsistent with a Poisson model. This was also true when the SCE data among mice implanted with BrdUrd tablets at ORAU and at BNL were combined (Table 9). In spite of the lack of a significant difference in mean SCE frequency between mice exposed to BrdUrd by these two methods, the dispersion of SCE was significantly elevated in tablet bearing mice over BrdUrd-infused mice ( P = 0.02). The biological basis for this increased dispersion in mice implanted with a BrdUrd tablet partially coated with paraffin over mice infused with BrdUrd probably lies in the differences in the time-dependent bioavailability of BrdUrd for incorporation into replicating DNA. The infusion system infuses BrdUrd at a constant rate into the peripheral blood of the recipient mouse (Schneider et al., 1978); thus, the level of BrdUrd in the circulatory system remains relatively constant across time beginning within - 3 0 min of the infusion period (Turturro et al., 1984). In animals implanted with an uncoated BrdUrd tablet, peak circulating BrdUrd levels are obtained - 4 h after tablet implantation, which then decreases over the next 5 h to essentially zero concentrations (King et al., 1982). Coating the tablet with agar (King et al., 1982) or partially coating the tablet with paraffin (McFee et al., 1983) lowers and broadens the peak availability of BrdUrd and incidentally lowers the frequency of SCE. However, even under these conditions, the circulating levels of BrdUrd fluctuate with time. Since SCE are induced by BrdUrd, either directly via incorporation or indirectly by affecting nucleotide pools (Schvartzman and Tice, 1982), it is likely that the increased hyperdispersion of SCE data in mice implanted with a BrdUrd tablet arises from the increased variability in circulating BrdUrd levels among the bone marrow cells. It would be expected that the multiple-injection design of Conner et al. (1978) would have more nearly mimicked the continuous infusion system than the BrdUrd tablet method. The intercellular distribution of SCE induced in vitro or in vivo by many genotoxic agents is hyperdispersed and inconsistent with a Poisson model (Rainaldi and Marriani, 1982; DuFrain, 1983; Whorton et al., 1984; Margolin et al., 1986).
35 TABLE 9 ANALYSIS OF SCE D A T A IN C O N T R O L M I C E Lab.
Method BrdUrd Admin.
Solvent
Number of mice
SCE frequency a Mean + SEM
H b Mean ± SEM
Number of exceptions c
Sum X2 d
df e
Critical X2 f
BNL
IV Infus.
PBS
28
4.76 + 0.217
0.97 _+0.065
2
655.1
672
733.1
BNL
SC Tablet
PBS
4
4.655:0.070
1.39+0.131
1
133.4 *
96
119.9
ORAU
SC Tablet
CO/PBS
16
5.015:0.193
1.27 + 0.116
4
362.5
384
430.4
BNL/ORAU
SC Tablet
CO/PBS
20
4.94-1-0.157
1.29+0.096
5
487.2
480
531.8
Abbreviations: BNL = Brookhaven National Laboratory; O R A U = Oak Ridge Associated Universities; I V = intravenous; SC = subcutaneous; CO = corn oil; PBS = phosphate-buffered saline. a Group mean S C E / c e l l frequency + the standard error of the mean among mice. b Group mean H value + the standard error of the mean among mice. c Numb er of animals where SCE data were significantly different from Poisson expectations. d Sum of Chi square values. e Degrees of freedom. f Critical Chi square value for a = 0.05. * Significantly different at a = 0.05.
While the biological basis for this increasing hyperdispersion with increasing mean SCE frequency has not been resolved, cell-to-cell differences in induced damage, proliferative capacity, cell stage sensitivity, DNA-repair activity a n d / o r available time for repair could modulate the distribution of SCE observed among exposed cell populations (Wolff et al., 1974; Sasaki, 1982; Sharer, 1982; Schvartzrnan and Gutierrez, 1980). For the in vivo study evaluated here, it was considered possible that chemical-specific differences in the pharmacokinetic distribution of reactive species among cycling cells in bone marrow could result in differences in SCE distribution which would not be readily apparent from an analysis of induced SCE frequencies. The 3 carcinogenic chemicals - - CP, DMBA, and MMC - - used in this study were selected for the evaluation of treatment time effects on SCE induction because of their wellestablished genotoxic activity (Preston et al., 1983; Latt et al., 1981) and their differing solubility and metabolic characteristics. The treatment times used were based on the two major approaches to treatment time selection used for in vivo SCE studies. The difference in approach is whether the time of chemical injection occurs around the onset of BrUrd exposure (e.g., Schneider et al., 1978), or around the beginning of the second cell cycle (e.g.,
Allen et al., 1977). The purpose of the former protocol was primarily to increase the opportunity for persistent D N A lesions to induce SCEs in more than one cycle (Tice and Schvartzman, 1982) and to provide time for reactive metabolites to be formed from slowly metabolized chemicals. The purpose of the latter protocol was to avoid the possibility that toxicity could remove heavily damaged cells from the proliferative pool before they had an opportunity to complete the necessary two S phases needed for SCE evaluation (Allen et al., 1977). Treatment of mice with CP and MMC (BNL) induced a significant increase in the dispersion of SCE in bone marrow cells in a dose-dependent manner. The increase in dispersion was also independent of treatment time. Treatment of mice with MMC at ORAU also increased the dispersion in SCE, but the increase was marginally nonsignificant. Comparative analysis by two-way ANOVA using common doses indicated no significant difference between the two laboratories in regard to the dispersion of SCE induced by MMC ( P = 0.78). However, since this analysis includes ~ data only at common doses, data at which the greatest increases in dispersion occurred were not evaluated ( > 2.5 m g / k g MMC). Because the distribution of individual animal SCE data at BNL
36 was different between M M C - t r e a t e d a n i m a l s infused with B r d U r d and those i m p l a n t e d with a B r d U r d tablet, an a d d i t i o n a l t w o - w a y A N O V A was c o n d u c t e d to c o m p a r e the + 1 h tablet d a t a from B N L with + 1 h tablet d a t a of O R A U . The dispersion d a t a were n o t significantly different between the two l a b o r a t o r i e s ( P = 0.12). The lack of a t r e a t m e n t - t i m e effect on the d i s p e r s i o n of S C E i n d u c e d by C P a n d M M C ( B N L ) suggests that there were no significant differences in the p h a r m a c o k i n e t i c d i s t r i b u t i o n of C P or M M C a m o n g different t r e a t m e n t times, no t r e a t m e n t time d e p e n d e n t v a r i a t i o n in cell-stage sensitivity, a n d no cellular toxicity to m o d u l a t e the response. T r e a t m e n t of mice with D M B A also h a d a significant effect on the intercellular d i s t r i b u t i o n of SCE within i n d i v i d u a l animals. However, as o p p o s e d to the o t h e r two chemicals, the increased / - / r e s p o n s e d e p e n d e d on t r e a t m e n t time, with the m a x i m u m H response occurring at the - 1 h t r e a t m e n t time a n d the m i n i m u m response occurring at the + 8 h t r e a t m e n t time. W h i l e the treatm e n t time effects could indicate d i s t r i b u t i o n a l differences in the levels of genotoxic m e t a b o l i t e s present in b o n e marrow, the differences in dispersion could also m e r e l y reflect the m a g n i t u d e of the i n d u c e d SCE yields (see Fig. 2). The d i s p e r s i o n of SCE i n d u c e d b y D M B A differed in o t h e r respects from the d i s p e r s i o n of SCE i n d u c e d b y C P a n d M M C . CP and M M C i n d u c e d a significant increase in H in a d o s e - d e p e n d e n t m a n n e r while the increase in d i s p e r s i o n following the t r e a t m e n t of mice with D M B A was n o t so strictly d e p e n d e n t on dose. Finally, at similar levels of SCE induction, the d i s p e r s i o n in mice treated with D M B A was m u c h greater than that o b s e r v e d in b o n e m a r r o w from mice treated with M M C , a n d to a lesser extent, CP. This difference in d i s p e r s i o n between a w a t e r - i n s o l u b l e c o m p o u n d a n d two water-soluble chemicals is p r o b a b l y d u e to p h a r m a c o k i n e t i c differences in m e t a b o l i s m a n d distrib u t i o n b a s e d on solubility characteristics. It is conceivable that some chemicals, due either to very limited d i s t r i b u t i o n or to highly specific cell stage specificity, m a y i n d u c e a significant increase in dispersion in the absence of a significant increase in m e a n S C E frequency. This analysis suggests that e x a m i n i n g the d i s t r i b u t i o n of SCE, in a d d i t i o n to m e a n frequency, is a useful m e t h o d for
e v a l u a t i n g agent specific p a t t e r n s in SCE induction.
Acknowledgements The a u t h o r s gratefully a c k n o w l e d g e the technical assistance of C. Luke, V. Miller, K. Lowe a n d A. W i l t o n a n d the p r e p a r a t i o n of the m a n u s c r i p t b y M. Rao. This research was s u p p o r t e d b y the National Toxicology Program under NIEHS lnteragency A g r e e m e n t s Y01-ES-20098 ( B N L ) a n d Y01-ES-20100 ( O R A U ) a n d was c o n d u c t e d in l a b o r a t o r i e s which are s u p p o r t e d b y the U.S. Dep a r t m e n t o f E n e r g y ; a c c o r d i n g l y , the U.S. G o v e r n m e n t retains a nonexclusive, royalty-free license to p u b l i s h o r r e p r o d u c e the p u b l i s h e d form of this c o n t r i b u t i o n , or allow others to do so for U.S. G o v e r n m e n t purposes.
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