- Email: [email protected]
Evaluation of the need for a late harvest time in the assay for chromosome aberrations in Chinese hamster ovary cells
Mutation Research, 292 (1993) 3-16 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1161/93/$06.00
3
MUTENV 08871
Evaluation of the need for a late harvest time in the assay for chromosome aberrations in Chinese hamster ovary cells Christian L. Bean and Sheila M. Galloway Merck Research Laboratories, W45-305, West Point, PA 19486, USA
(Received 19 October 1992) (Revision received 15 December 1992) (Accepted 29 January 1993)
Keywords: Chromosome aberrations; CHO, in vitro; Kinetics; Sampling time; Clastogens
Summary Harvest time is one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. In CHO cells we have found that for a variety of chemicals one harvest time near 20 h is optimal following a 3-h treatment (Bean et al., 1992). However, some guidelines for testing for regulatory purposes recommend an additional late harvest time 24 h after the first. We tested 10 diverse chemicals in CHO-WBL cells harvested 20-21 h and 42-44 h from the beginning of a 3-hr treatment. We added BrdUrd after treatment and recorded the total% of aberrant cells, and the proportions of aberrations (abs) in first (M1), second (M2) or later metaphases. The chemicals fell into 3 categories: ab yield greatly decreased at 44 h: benzo[a]pyrene, cadmium sulfate, chlorambucil, 2,6-diaminotoluene, 4-nitroquinoline N-oxide and mitomycin C (e.g., 37.0% cells with abs at 20 h and 1.0% at 44 h); ab yields similar at 20 and 44 h: 2-aminobiphenyl, eugenol and 8-hydroxyquinoline (e.g., 8.5% at 20 h and 7.0% at 44 h); and one, dimethylnitrosamine (DMN), which was detected at both times but gave a stronger response at 44 h than at 20 h (e.g., at 10 mM: 6.2% at 20 h and 25.0% at 44 h). This DMN effect was not seen in normal diploid human cells. For DMN the higher ab levels at 44 h than at 20 h were contributed by abs in M3 cells. Thus, while for some chemicals ab yields decrease with successive division, further increases can be seen in CHO in later metaphases, notably for DMN. Overall, however, after a 3-h pulse treatment of CHO cells a positive ab result could be obtained at the early harvest time (20 h) for all 10 chemicals.
The time at which metaphase cells are sampled after treatment is critical for detection of chromosomal aberrations (abs). Since abs induced by most chemicals are produced during
Correspondence: Christian L. Bean, Merck Research Laboratories, W45-305, West Point, PA 19486, USA.
DNA replication (Evans and Scott, 1969), the harvest time must be selected to allow cells to progress through S-phase after treatment. Based on studies of abs induced by ionizing radiation it was clear that abs should be scored in the first metaphase after they are formed to avoid loss by cell death during mitosis or conversion of the initial abs into complex derivatives during subsequent cell cycles (Carrano and Heddle, 1973; Das
and Sharma, 1987). Thus, a sampling time of no more than 1 cell-cycle length from the beginning of treatment has been used, e.g., about 10 h from the beginning of a 3-h pulse treatment in CHO ceils which have a cell-cycle length of 12-14 h (Galloway et al., 1985). Because cell-cycle delay often results from clastogen exposure, that protocol was modified to allow delayed cells time to progress to mitosis; sampling times of, e.g., 18-28 h have proved appropriate in CHO cells (e.g., Galloway et al., 1987; Loveday et al., 1989). There are ongoing international discussions of appropriate harvest times for standard testing protocols for in vitro chromosome ab assays. The UKEMS guidelines (Scott et al., 1990) recommend a first harvest at about 1.5 cell-cycle lengths from the beginning of treatment, with a second harvest 24 h later, in cases where negative or equivocal results are seen at the first sampling time. Japanese guidelines (1990) recommend both 24 and 48 h sampling times, but these harvests are done after continuous treatment for 24 or 48 h in contrast to our current protocol with a 3-h treatment. We reported previously that for a variety of chemicals a single harvest time about 17-21 h after the beginning of a 3-h pulse treatment was adequate for ab detection when doses were appropriately selected (Bean et al., 1992). In a study of V79 hamster ceils Thust et al. (1980) found maximal ab yields occurred at widely different times (e.g., 24-60 h) after a 1-h pulse treatment even among a group of closely related chemicals. Here we explore further the chromosome ab kinetics for 10 chemicals by harvesting at 20-21 and 42-44 h from the beginning of a 3-h treatment. Few chemicals other than some nitroso compounds in Chinese hamster V79-E cells (Thust et al., 1980; Thust, 1982) and nitrogen mustard and maleic hydrazide with plant ceils (Evans and Scott, 1964, 1969) have been investigated in this way. Some of the chemicals and concentrations in the present study give weak or equivocal responses. The chemicals vary widely in their modes of action and include alkylating and crosslinking agents, a metal salt and a catechol. Abs were recorded in cells labelled with 5bromodeoxyuridine to determine how many cycles each metaphase cell had undergone since treatment.
Materials and methods
Culture of CHO WBL cells CHO cells were maintained in McCoy's 5A medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 2 mM L-glutamine, 100 U / m l penicillin and 100 /zg/ml streptomycin (all Gibco). Cultures were incubated at 37°C in a humidified, 5% CO 2 atmosphere. Metabolic activation system Liver homogenate ($9 fraction) was prepared from phenobarbital//3-napthoflavone treated male Sprague-Dawley [CRCD Crl:CD(SD)BR] rats from Charles River Breeding Farms, Raleigh, NC. $9 was stored at -70° C to -80°C and thawed immediately before use. $9 was mixed with sodium NADP (Boehringer Mannheim), and trisodium isocitrate (Sigma) in serum-free medium immediately before use. The final concentrations in cultures were: $9, 15 /xl/ml; NADP, 0.8 m g/ m l (1.05 mM) and trisodium isocitrate, 1.5 mg/ml (5.8 mM). Test chemicals and solvents 2-Aminobiphenyl (2-ABP; Cas No. 90-41-5), benzo[a]pyrene (BP; CAS No. 50-32-8), cadmium sulfate (CdSO4; CAS No. 10124-36-4), chlorambucil (CAB; CAS No. 305-03-3), dimethylnitrosamine (DMN; CAS No. 62-75-9), dimethyl sulfoxide (DMSO), 8-hydroxyquinoline (8-HQ; CAS No. 148-24-3), Mitomycin C (MMC; CAS No. 50-07-7) and 4-nitroquinoline N-oxide (4NQO; CAS No. 56-57-5) were from Sigma, St. Louis, MO. 2,6-Diaminotoluene (2,6-DAT; CAS No. 823-40-5), and eugenol (EUG; CAS No. 9753-0) were from Aldrich. Deionized, distilled water (dH20) was purchased from Gibco. CdSO4, DMN and MMC were prepared in dH20.2-ABP, BP, 2,6-DAT, EUG and 8-HQ were prepared in DMSO. 2-ABP, BP, DMN, EUG and 8-HQ were tested in the presence of $9 metabolic activation. None of the chemicals changed the pH of the medium substantially as judged by the color of the indicator. Assessment of chromosome-aberration kinetics About 24 h before treatment, 1.2 x 106 cells were seeded in 10 ml medium in 75-cm 2 flasks
(Corning Glass Works, Corning, NY). Just before treatment, the medium was replaced. For tests without $9, 9.9 ml of complete medium was added. For tests with $9 mix, cultures were rinsed once with prewarmed (37°C) Dulbecco's phosphate-buffered saline without Ca z+ and Mg 2+ (DPBS, Gibco) and refed with 9.9 ml of serumfree medium containing the $9 metabolic activation system. Test compound (100 ttl) was added from concentrated stocks (100 × ), and cultures both with and without $9 mix were incubated for 3 h at 37°C, then washed twice with warmed DPBS and refed with 10 ml of prewarmed complete medium containing 10 /~M 5-bromo-2'-deoxyuridine (BrdUrd, Sigma). BrdUrd was present during the 3-h treatment with BP, but not in later experiments, during treatment with all other chemicals. Cultures were harvested at 20 and 44 h (or 21 and 42 h for benzo[a]pyrene) from the beginning of treatment, and 2.5-3 h after addition of 0.1 /zg/ml colcemid (Gibco). Cells were harvested by mitotic shake-off, or by trypsinization to allow cell counts (Coulter counter) for cytotoxicity estimation. Cells were treated with hypotonic KCI (75 raM) for 1-3 rain at room temperature, washed twice with fixative (methanol:glacial acetic acid, 3:1 v/v), dropped onto slides and air-dried. Slides were stained for sister chromatid differentiation by the fluorescence plus Giemsa technique (Perry and Wolff, 1974) as modified by Goto (1978).
Toxicity assesssment Toxicity was assessed by estimates of monolayer confluence and observations of abnormal cell morphology, or by Coulter cell counts of trypsinized cultures. Estimates of confluence were generally within 10% of Coulter cell counts when both measurements were made.
Dose selection The maximum concentrations to be tested were based on our previous experiments in CHO cells by the same protocol and generally did not exceed 50% toxicity, based on reductions of cell counts at 20 h compared with concurrent controls. Lower doses were also selected based on previous experiments and were chosen to yield
weak to moderate effects. The maximum concentration tested was less than 10 mM except for CAB, DMN and 2,6-DAT which were tested up to 20 mM, 18 mM and 20 mM, respectively, due to lack of cytotoxicity. The osmolality of the medium was not increased over control medium with solvent.
Metaphase classification Abs were scored in cells classified in the following categories: M1, even, dark staining on both chromatids. M1 + , 1 partial S phase in BrdUrd followed by 1 complete S phase: regions with sister-chromatid differentiation (SCD), and regions with both chromatids still dark. These cells are in their second metaphase since treatment. M2, 2 complete S phases in BrdUrd: full SCD. M2 + , 1 partial S phase in BrdUrd followed by 2 complete S phases: regions with SCD and regions with even, light staining on both chromatids. These cells are in their third metaphase since treatment. M3, 3 complete S phases in BrdUrd: about three-quarters of the chromatin appears evenly, light stained with the remainder dark.
Aberration scoring At 20 h, 200 cells per point (100 M1 and 100 M1 + , where possible) were scored for abs unless there were very high ab frequencies. At 44 h, we scored 100 or 200 consecutive cells for abs, and recorded the division each cell was in, e.g., M1, M2. We used metaphases with 19-23 chromosomes, the modal number being 21. All types of structural abs were recorded; chromatid and isochromatid gaps were recorded but not ineluded in the totals of abs. We defined a gap as an achromatic region equal to or smaller than the width of a chromatid in that cell, or a larger lesion with visible connecting material across the gap. We calculated both the percentage of aberrant cells (the number of cells with structural abs per 100 cells) and the frequency of abs (the total number of abs per 100 cells), since a cell may have more than one ab. Ceils with 10 or more abs were classed as severely damaged (SD) cells, and scored as one aberrant cell but as 10 abs.
Mitomycin C
Benzo(a) pyrene
~ 6 0 1~--]21 HR ~ 4 2 HR
~.70 E~20 HR ~ 4 4 HR
"
~ 5o ~ 4o
~3o l- 20
~ 20
,<10
~1o <
0
0.35/JM
0.50 ,uM
"
0
1.00 pM
20 pM
Dose
Cadmium
8
40/JM
Dose
2,6-Diaminotoluene
Sulfate
T E~]20 HR ~ 4 4 HRJ
~20 "o
"k
~15 -o
~-
-
.
1~20 HR [ ] 4 4 HR I
8
7 15
7 ~I0
_.m - -
.
E 5
<
<
°~ o
1.0/JM
~
1,5/JM
~
o
10 mM
Dose
4-Nitroquinoline
~30
14 mM
18 mM
Dose
4-Nitroquinoline N-oxide
N-oxide
~ 3 0 r~]20 HR [ ] 4 4 HR
~]20 HR [ ] 4 4 HR
.k
o 25
=o25
-o
~-- 2 0
~C2o
815
81s
=o)
c .~10
~r
~s 0
o~ o 0.25/JM
0.50/JM
1 O0 ,uM
Chlorambucil
o 25 "1o
.
.
.
.
.
.
.
.
.
J .
_ ,\\\ I\\\11 iX\\ll r\\\
....
8 15 clO
0
5 mM
10 mM Dose
1.00 uM
Dose
Dose
A 3 0 1%--]20 HR [ ] 4 4 HR
J
20 mM
2.50 ,uM
For selected points, the percentage of aberrant cells in treated cultures was compared to concurrent controls by the "normal test" of Margolin et al. (1983), a version of the Chi square test based on a standard normal approximation. The following abbreviations are used in the text and figures: TD, chromatid deletion, TE, chromatid exchange, CD, isochromatid (chromosome) deletion and CE, chromosome exchange. Results Controls The mean percentage of cells with abs for negative and solvent controls was 2.5% (range 0.5-6.0%, 11 determinations). Concurrent control percentages were subtracted from treated values for all data shown in the figures. Aberration yields at two harvest times The chemicals can be grouped into 3 categories with regard to ab responses at the two harvest times. Those in Group I (BP, CdSO4, CAB, 2,6-DAT, 4-NQO and MMC) had substantial numbers of abs at 20 h but few abs remained at 44 h (Fig. 1). The lower ab yield observed at 20 h for 40 ~M BP than for 20 ~ M BP may be the result of differing degrees of cell cycle delay at these 2 doses such that the maximum yield of aberrations at 40 tzM BP might not be reached by 20 h. For 3 clastogens, 2-ABP, EUG and 8-HQ (Group II) the ab increases at 20 h were similar to those at 44 h (Fig. 2a). Similar results were observed in a repeat experiment. One chemical, DMN (Group III), was positive both at 20 and at 44 h but gave a much stronger response at 44 h (Fig. 2b). Distribution o f aberrations over successive cell cycles Cell-cycle delay seen from % M1 and M1 + ceils at 20 h is shown in Table 1. Control cultures had substantial numbers of M1 + cells at 20 h,
i.e., 62-83%. For the Group I chemicals, abs were scored at doses that yielded greater than 60% survival (cell counts or monolayer confluence) compared with controls at 20 h. The distribution of abs across division stages was comparable at all dose levels, so the cells scored were pooled across dose levels for the following analyses. Most of the abs seen for Group I chemicals at 20 h were in M1 cells (Table 2). Where second metaphase (M1 + ) cells were seen at 20 h, few had abs. At 44 h, the few abs present were observed in all divisions, M1 through M3, though M3 cells generally had fewer abs than M2 cells. For Group II chemicals (similar yields at 20 and 44 h) abs at 20 h were predominantly in M1 cells; very few more advanced cells were seen (Tables 1 and 2), due to cell-cycle delay. Quite toxic doses were required to induce abs for these Group II chemicals, i.e., cell counts were reduced to about 50% of controls. At 44 h (Table 2) 20-50% of the few remaining M1 cells contained abs (e.g., 2-ABP 3 ab cells/14; EUG, 10 ab cells/21) in contrast to 6-14% of the M1 cells at 20 h. Thus, there was a tendency for the extremely delayed M1 cells found at 44 h to contain abs. For 2-ABP and EUG, a reduction in abs with successive divisions was seen (Table 2): overall percentages of aberrant cells were lower in second than in first division cells (e.g., EUG, 16.7% of 48 M2 cells el. 50% of 21 M1 cells) and lower still in third division cells (EUG, 7.8% of 231 M3 cells). These observations were confirmed in repeat experiments (data not shown). However, for 8-HQ the abs at 44 h were all in third division cells (8.2% of 195 M3 cells); few earlier division cells were observed (Table 2). DMN yielded abs at 20 h in both M1 (7.3% of 300) and M1 + (second division) cells (4.6% of 373, P = 0.03 cf. control of 1.3%). Most of the abs observed at 44 h were in third division cells (17.0% of 276 M3 cells) but a higher percentage of aberrant cells was found in the few second division cells observed (69.6% of 23 M2 cells).
Fig. 1. Group I chemicals. Chromosome aberration induction at 20 or 21 h and 42 or 44 h after a 3-h treatment in CHO cells. Control rates have been subtracted from data shown. 200 cells scored per point at 20 or 21 h except when rate was over 30%, 100 cells scored, or over 50%, 50 cells scored. 100 cells scored at 42 or 44 h. * indicates a statistically significant ( P < 0.05) increase over controls.
a 8-Hydroxyquinoline
Eugenol
~r
~ 1 0 ["~20 HR [ ] 4 4 HR
~2
8 o~
81
o
~ 4
c #_
<
< 1.2 mM
1.4 mM
1.6 mM
:iFJ 40 pM Dose
Dose
50 #M
2-Aminobiphenyl ~ 1 0 [X]20 HR ~ 4 4 HR
7
*
8
*
O.O mM
1.0 mM
Dose
b Dimethylnitrosamine ~ 4 0 [ ] 2 0 HR [ ] 4 4 HR /J
~ao 82o
.~I0 <
~ X
0
5 mM
10 mM
20 mM
Dose
Fig. 2. Chromosome aberration induction at 20 and 44 h after a 3-h t re a t me nt in C H O cells. Control rates have been subtracted from data shown. 200 or more cells scored per point at 20 h, 100 cells scored at 44 h. * indicates a statistically significant ( P < 0.05) increase over controls. (a) Group 1I chemicals. At 20 h for 1.6 mM E U G only 114 cells were available. (b) G roup I l l chemical.
Types of aberrations
primarily chromatid deletions (TD) and exchanges (TE; Fig. 3). Data shown are for the highest dose only since similar distributions CdSO4, were
At 20 h the abs scored for 5 of the 6 Group I chemicals, MMC, 2,6-DAT, CAB, 4-NQO and
TABLE 1 C E L L C Y C L E D E L A Y IN C H O C U L T U R E S Treatment
Dose
Percentage of cells per division 20h M1
44 h M1 +
M1
M2
M3
8
92
1 1
11 22 30
89 77 69
1
22 44
77 55
1
4 4 1
96 96 98
1
50 65
50 34
4 5
96 95 99
Group I Negative control a MMC
CdSO 4
2,6-DAT
BP CAB
4-NQO
17
83
0.35/~M 0.50/xM 1.00/xM
87 100 b 100
13
1.00 ~ M 1.50 ~ M
70 100
30
10.00 m M 14.00 m M 18.00 m M
19 43 40
81 57 60
20.00/~M 40.00/xM
100 100
5.00/x M 10.00/IM 20.00/xM
80 81 100
20 19
0.25/~M 0.50/~M 1.00/xM 2.50/xM
26 70 69 86
74 30 31 14
5
100 99 100 95
39
62
2
98
29
71
1 1
Group H Negative control a Solvent control
2
98
EUG
1.2 m M 1.4 m M 1.6 m M
100 100 100
8 2 11
13 12 23
79 86 66
2-ABP
0.9 m M 1.0 m M
100 100
9 5
34 59
57 36
40.0/~M 50.0/~M
99 95
1 5
2 3
98 97
20
80
2
98
40 30 44
60 70 56
1 5 17
99 95 82
a
8-HQ
Group III Solvent control a DMN
5 mM 10 m M 20 m M
a Representative control data since several experiments were done. b Predominantly M1 cells were observed.
1
10
were seen at all doses. In addition, 2,6-DAT induced severely damaged (SD) cells containing chromatid deletions and exchanges. The other Group I chemical, BP, yielded chromatid exchanges and isochromatid deletions (CD) at 21 h (Fig. 3). These abs were in M1 ceils since no more advanced ceils were available due to cellcycle delay or cell death. By 44 h, few abs remained for any of these 6 chemicals. For the Group II chemicals the percentages of cells with abs at 20 h were similar to those at 44 h (Fig. 2a). Chromatid deletions and exchanges and
isochromatid deletions were observed at 20 h for 2-ABP (data not shown since too few M3 cells were available) and for E U G (Fig. 4). At 44 h, for 2-ABP chromatid exchanges were seen in the few M1 ceils observed and isochromatid deletions were observed in M2 and M3 cells (data not shown since too few M3 cells were available, Table 2). For EUG, by 44 h abs were chromatid exchanges, isochromatid deletions and chromosome exchanges (CE) in M3 ceils. Some chromatid exchanges were found both in the few M1 cells, and in M3 cells (Table 2, Fig. 4). However,
TABLE 2 ANALYSIS OF ABERRANT CELLS BY METAPHASE CATEGORY Treatment
Dose
20 h
44 h
% ab
ab cells/cells scored
cells
M1
M1 +
% ab cells
ab cells/cells scored
M1
M2 a
M3 b
1/8
1/92
0/1 1/1
1/11 1/22 1/30
4/89 0/77 3/69
2/2
3/63
7/235
1/1
0/22 3/44
0/55
1/1
3/66
1/133 4/96 1/96
1/1
0/4 0/4 0/1
1/1
0/9
10/290
0/2
6/100 10/131
1/100 1/67
0/2
16/231
2/167
Group I Negative control c
MMC
0.35/zM 0.50/zM 1.00/zM
3.5
5/100
2/100
2.0
9.0 37.0 62.0
16/100 37/100 31/50
2/100 NA NA
5.0 1.0 5.0
84/250
2/100
5/100 36/200
2/100 NA
41/300
2/100
13/100 17/100 26/100
4/100 3/100 1/100
56/300
8/300
51/100 38/100
NA NA
Totals
CdSO4
1.00 /zM 1.50/zM
3.5 18.0
Totals
2,6-DAT
10.00 mM 14.00 mM 18.00 mM
8.5 10.0 13.5
Totals
BP d
20.00/zM 40.00/xM
51.0 38.0
5.00/~M 10.00/~M 20.00/zM
6.0 15.5 31.0
Totals
4-NQO
0.25 ~M 0.50 tzM 1.00/~M 1.00/~M 2.50/xM Totals
4.0 1.0 6.0
3.5 5.5
89/200
Totals
CAB
1.0 6.0
2.0 3.5 8.5 3.9 27.5
9/100 27/100 31/100
3/100 4/100 NA
67/300
7/200
2/100 5/100 15/100 10/100 57/200
2/100 2/100 2/100 0/156 6/29
89/600
12/485
5/98
0/4 2/5 0/1
7,/96 7/95 9/99
2/10
23/290
0/1
6/100 5/99
3/3
1/10 19/97
10/190 4/100
3/3
20/108
33/589
7,0 9.0 9.0
6.0 5.0 8.0 5.5 13.0
1/78
8/100
11 TABLE 2 (continued) Treatment
Dose
20 h % ab cells
44 h ab cells/cells scored M1 M1 +
% ab cells
ab cells/cells scored M1 M2 a
M3 b
Group H
Negative control c DMSO solvent control c EUG
1.0% 1.2 mM 1.4 mM 1.6 mM
2.5
5/100
0/100
2.0
0/2
2/98
0.5
0/100
1/100
1.0
0/2
1/98
7.0 7.0 14.0
14/200 14/200 16/114
Totals 2-ABP
0.9 mM 1.0 mM
40.0 ~M 50.0 tzM
10.0 6.0 20.0
44/414 8.0 6.0
Totals 8-HQ
NA NA NA
16/200 12/200
NA NA
1.0 7.0
28/400 6.4 8.1
Totals
13/200 17/200
0/2 0/10
30/400
0/12
5/8 2/2 3/11
1/13 1/12 6/23
4/79 3/86 11/66
10/21
8/48
18/231
0/9 3/5
0/34 3/59
1/57 1/36
3/14
3/93
2/93
0/2 0/3
5/98 11/9"
0/5
16/19
5.0 11.0
Group III
dH20 solvent control DMN
1%
2.3
4/100
2/159
3.5
0/5
5 mM 10 mM 20 mM
4.5 6.2 6.6
7/100 9/100 6/100
3/121 6/141 8/111
5.0 25.0 34.0
1/1
0/1 3/5 13/17
5/99 22/95 20/82
1/ 1
16/23
47/276
Totals
22/300
17/373
7/195
This category includes a small number of M1 + cells which although intermediate in staining, are in their second metaphase since treatment. b This category includes M2+ cells which although intermediate in staining, are in their third metaphase since treatment. c Representative control since several experiments were done. d Harvest times for BP were 21 and 42 h after dosing. NA Few or no M1 + cells available for analysis. The data in this table are absolute values and have not been corrected for control rates. The control range was 0.5-6.0%, mean 2.47%, 11 determinations. Where possible, 100 M1 and 100 M1 + cells were scored at 20 h, while at 44 h 100 or 200 consecutive cells were scored. a
at 1.6 m M in a r e p e a t e x p e r i m e n t with E U G , in which there was slightly g r e a t e r toxicity, the chrom a t i d exchanges were seen in M1 a n d M2 cells at 44 h. This illustrates how ab types can vary from o n e e x p e r i m e n t to the next d u e to slight c h a n g e s in cell-cycle kinetics, i.e., at 1.6 m M , 66% of the cells were M3 at 44 h in the first e x p e r i m e n t b u t only 30% were M3 in the second. Some of the
M1 cells r e c o r d e d at 44 h were severely d a m a g e d ( 6 / 2 1 M1 cells) b u t 11 o t h e r M1 cells f o u n d had n o visible d a m a g e ( T a b l e 2). I n contrast to the 44-h result, n o SD cells were observed at 20 h for E U G . T h u s with E U G , the heavily d a m a g e d (SD) cells were greatly delayed, a p p e a r i n g as M1 cells at 44 h. I n contrast, SD cells with 2 , 6 - D A T were seen at 20 h a n d were gone by 44 h. F o r 8-HQ,
12 M i t o m y c i n C - 1.0 p M
!I
r~]20HR- M1 ~ 4 4 H R - M 3
g 7O 6o
~4o
~30 ~'20 ~ u. 0
2 , 6 - D i a m i n o t o l u e n e - 18 m M
o
-10 g-
~s g SD
Data from 50 M1 cells and 69 M3 cells
Cadmium
~ 14
.
.
u_ 0
TD
Benzo(a)pyrene
S u l f a t e - 1.5 p M
.
.
TE CD CE Aberration Types
SD
Data from 100 M1 cells and 98 M3 cells
~50/~
[~20HR-M1 [ ] 4 4 H R - M 3 .
~44HR'M~
-~15
TE CD CE Aberration Types
TD
I~20HR'M1
8 20
.
- 20 pM
11~]21HR-M1 D 4 2 H R - M 3 J
-
__.=12
='40
v
8 ao
8 8
~6
4 u_ 0
4- 0 TD
TE CD CE Aberration Types
TD
SD
TE CD CE Aberration Types
......-~ SD
Data from 100 M1 cells and 100 M3 cells
Data from 200 M1 cells and 55 M3 cells
Chlorambucil - 20/JM
4-Nitroquinoline N-oxide - 2.50/3M /
= 35/'~
[ ] 20 HR-M1 [ ] 4 4 H R - M 3 1
3
5
/
~
-
M
3
J
~ 3o ,4, ~ 25 .I 8 20 4"
o-
~3o
\\
8 2o
~ 25
\\ \\ \\ \\
5
u_ 0
- • \\ \\ \\
\\
TD
TE CD CE Aberration Types
SD
TD
TE CD CE Aberration Types
SD
Data from 200 M1 cells and 100 M3 cells Data from 100 M1 cells and 99 M3 cells Fig. 3. Types of aberrations induced at 20 and 44 h after a 3-h treatment without $9, except BP, with $9. The following abbreviations are used: TD, chromatid deletion, TE, chromatid exchange, CD, isochromatid (chromosome) deletion, CE, chromosome exchange and SD, severely damaged cells, scored as one aberrant cell but as 10 abs. Control frequencies have been subtracted from each data point.
13
8 - H y d r o x y q u i n o l i n e - 50 pM
Eugenol - 1.6 mM ~15 ca
o°10
g
~. 5
== u. 0
TD
TE
CD
CE
SD
TD
Aberration Types
TE
CD
Aberration
Data from 114 M1 cells and 66 M3 cells
CE
SD
Types
Data from 200 M1 cells and 97 M3 cells
Dimethylnitrosamine - 20 mM
D
o=
o
L,
TD
TE
CD
CE
SD
Aberration Types Data from 100 M1 cells and 82 M3 cells
Fig. 4. Types of aberrations induced at 20 and 44 h after a 3-h treatment with S-9. The following abbreviations are used: TD, chromatid deletion, TE, chromatid exchange, CD, isochromatid (chromosome) deletion, CE, chromosome exchange and SD, severely damaged cells, scored as one aberrant cell but as 10 abs. Control frequencies have been subtracted from each data point.
the abs at 20 h were mainly chromatid deletions and exchanges in addition to some SD cells (Fig. 4). By 44 h, all abs were in M3 cells and these were primarily chromosome exchanges in addition to a few chromatid exchanges at 50 /xM. There were no SD cells at 44 h for 8-HQ. Thus, overall for the Group II chemicals there was loss of severely damaged cells through division, and derived types of abs were seen at later divisions (e.g., chromosome exchanges, CE). Most of the CE observed in later divisions were not associated with acentric fragments (AF) (e.g., 8HQ: 3 A F / 2 2 CE; EUG: 3 AF/21 CE). For DMN the ab increases at 20 h were low but significant, e.g., totals of 6.2% at 10 mM and 6.6% at 20 mM (P < 0.02 for both dose levels
compared with controls; Fig. 2b shows percentages with controls subtracted). The abs included chromatid deletions and exchanges and isochromatid deletions (Fig. 4). By 44 h the abs were mainly chromatid exchanges and isochromatid deletions. Some SD cells were scored at 44 h in M2 cells (not shown) but most of the abs at 44 h were in M3 cells. DMN is the only chemical for which SD cells appeared as cells beyond the first metaphase. Discussion
A general practice has been to score abs in the first metaphase following treatment, to detect primary abs (Evans and O'Riordan, 1975; Preston
14 et al., 1981). Abs can lead to cell death due to mechanical difficulties at anaphase a n d / o r loss of material in the daughter cells. Also, some abs may lead to derived-type abs such as dicentrics or rings (reviewed by Savage, 1975). Thus, scoring abs in cells other than first division metaphases could lead to erroneous estimates of clastogenic potential. Careful selection of harvest time is thus one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. In previous work with the C H O cell system, sampling times used to detect abs were 9.5-12 h and were extended to 18-28 h if there was evidence for substantial cell-cycle delay from the numbers of first- and second-division metaphases in preliminary sister-chromatid exchange assays (Galloway et al., 1985, 1987; Ivett et al., 1989; Loveday et al., 1989). More recently, we found that, following a 3-h treatment, one harvest time near 20 h was optimal for obtaining positive results in C H O cells for a range of chemicals (Bean et al., 1992). However, some published and proposed guidelines recommend an additional very late harvest time (24 h after the first) for chemicals that are equivocal or negative at a first sampling time about 1.5 cycle times from the beginning of treatment (Scott et al., 1990). H e r e we tested 10 diverse chemicals and found that after a 3-h pulse treatment of C H O cells a single harvest time about 20 h after the beginning of treatment was adequate for ab detection, although one of the 10 chemicals had a greater yield of abs at 44 than at 20 h. Three categories were observed: ab yields greatly decreased at 44 h: BP, CdSO4, CAB, 2,6-DAT, 4 - N Q O and MMC; ab increases detected equally at 20 and 44 h: 2-ABP, E U G and 8-HQ, and one chemical, DMN, that was detected at both times but gave a much stronger response at 44 h, mainly in M2 to M3 cells. For the G r o u p I chemicals few abs were detected at 44 h probably because most of the clastogenic damage was lethal. Such toxicity is consistent with results for two of these chemicals (MMC and CdSO 4) which showed little initial toxicity at 3 or 10 h after treatment but dramatic reductions in cell counts and A T P content by 24 h (Armstrong et al., 1992). Colony-forming ability
measured at 7 - 8 days was also reduced at doses that caused large numbers of abs i.e., 1.67 g M CdSO 4 and 1.0/xM MMC. In contrast, significant toxicity was observed immediately after the 3-h treatment with 2-ABP, E U G and 8-HQ at clastogenic dose levels (Armstrong et al., 1992). Since we added BrdUrd after treatment we were able to record the percentage of aberrant cells and the total number of abs in first (MI), second (M2) or later metaphases. The chromosome exchanges in later division cells seen with E U G and 8-HQ suggests conversion to derived types of abs with successive divisions. For D M N the higher ab levels at 44 h than at 20 h were contributed by M3 cells, and apparently were arising in the second and third cycles after treatment possibly as a result of long-lived lesions not expressed as chromosome abs through replication or repair in the first cell cycle. The presence of some chromatid exchanges in M3 cells with E U G suggests persistence of lesions induced by E U G also. Chromosomal effects of long-lived lesions have been observed for other chemicals in some cell types. In plant cells (Vicia faba root tips), Evans and Scott (1969) found that after treatment with nitrogen mustard in S phase the yield of abs was significantly higher at the second mitosis than at the first mitosis. Similar results had been reported for maleic hydrazide (Evans and Scott, 1964). In Chinese hamster cells, Ikushima and Wolff (1974) also found evidence for long-lived lesions that could induce chromosome abs during first, second or third replications following irradiation of G~ cells with X-rays or UV light. For a series of nitroso compounds (Thust et al., 1980) maximal ab increases occurred 24-60 h after l-h pulse treatments of Chinese hamster V79-E cells (doubling time of 12-14 h). For DMN, Thust (1982) observed a maximal yield of abs at 36 h after a 3-h treatment in V79-E cells. At 32 h there were chromatid exchanges and to a lesser extent chromatid breaks, but few chromosome exchanges. These observations by Thust (1982) are in agreement with our data for D M N in C H O cells. In both labs, while the maximum response was later, significant increases in abs were found as early as 20-24 h. In addition, Natarajan et al. (1976) found increases in abs with D M N in C H O
15
cells as early as 15 h after a one-hour treatment, but found much higher yields of abs at 24 h. Studying sister-chromatid exchanges (SCEs), Wolff (1978) found that when CHO cells synchronized in G 1 were exposed to ethyl methanesulfonate, methyl methanesulfonate, 4-nitroquinoline- 1-oxide or acetoxyacetylaminofluorene, long-lived lesions were produced that led to the formation of SCEs in the first, second and to a lesser degree third cell cycle after treatment. Similar results were reported by Linnainmaa and Wolff (1982) for angelicin, the monofunctional derivative of 8-methoxypsoralen, in combination with UVA light. We are examining further the occurrence of abs induced by some chemicals in first, second, third or later division cells. The persistence of lesions could be specific to the cell type, probably related to repair capacity and/or the stringency of control mechanisms allowing cell cycle progression to metaphase in the presence of genetic damage (Kung et al., 1990). Our preliminary results show that in contrast to the increase in abs with time in CHO ceils, the ab yield with DMN decreases with successive cell generations in freshly isolated human lymphocytes and in early passage normal human fibroblasts. When cells are harvested about 1.5 cell cycles after treatment the initial yield of abs in human cells is similar to or less than that in CHO cells but direct comparisons are difficult since optimal ab yields may vary due to slight differences in cell kinetics. Our preliminary work suggests an increase with time may be specific to chemical agents (certain alkylating agents) and cell types (may not occur in untransformed human cells). We are investigating the potential role of O6-alkylguanine-DNA alkyltransferase in the time course of ab production by alkylating agents. From the present study we conclude that after a 3-h treatment of CHO cells a 20-h harvest time effectively detected clastogenic chemicals and a later harvest time was not required even for chemicals that are difficult to detect as clastogens. Acknowledgements We thank Timothy Johnson for his excellent technical contributions.
References Armstrong, M.J., C.L. Bean and S.M. Galloway (1992) A quantitative assessment of the cytotoxicity associated with chromosomal aberration induction in Chinese hamster ovary cells, Mutation Res., 265, 45-60. Bean, C.L., M.J. Armstrong and S.M. Galloway (1992) Effect of sampling time on chromsome aberration yield for 7 chemicals in Chinese hamster ovary cells, Mutation Res., 265, 31-44. Carrano, A.V., and J.A. Heddle (1973) The fate of chromosome aberrations. J. Theoret. Biol., 38, 289-304. Das, B.C., and T. Sharma (1987) The fate of X-ray-induced chromosome aberrations in blood lymphocyte culture, Mutation Res., 176, 93-104. Evans, H.J., and M.L. O'Riordan (1975) Human peripheral blood for the analysis of chromosome aberrations in mutagen tests, Mutation Res., 31, 135-148. Evans, H.J. and D. Scott (1964) Influence of DNA synthesis on the production of chromatid aberrations by X-rays and maleic hydrazide in l/icia faba, Genetics, 49, 17-38. Evans, H.J., and D. Scott (1969) The induction of chromosome aberrations by nitrogen mustard and its dependence on DNA synthesis, Proc. Roy. Soc. B, 173, 491-512. Galloway, S.M., A.D. Bloom, M. Resnick, B.H. Margolin, F. Nakamura, P. Archer and E. Zeiger (1985) Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: comparison of results for 22 compounds in two laboratories. Environ. Mutagen., 7, 1-51. Galloway, S.M., M.J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A.D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J. Rimpo, B.H. Margolin, M.A. Resnick, B. Anderson and E. Zeiger (1987) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals. Environ. Mol. Mutagen., 10, Suppl. 10, 1-175. Goto, K., S. Maeda, Y. Kano and T. Sugiyama (1978) Factors involved in differential giemsa-staining of sister chromatids, Chromosoma, 66, 351-359. Ikushima, T. and S. Wolff (1974) UV-induced chromatid aberrations in cultured Chinese hamster cells after one, two, or three rounds of DNA replication, Mutation Res., 22, 193-201. Ivett, J.L., B.M. Brown, C. Rodgers, B.E. Anderson, M.A. Resnick and E. Zeiger (1989) Chromosome aberrations and sister chromatid exchange tests in Chinese hamster ovary cells in vitro, IV. Results with 15 chemicals, Environ. Mol. Mutagen., 14, 165-187. Japanese Guidelines for Toxicity Studies of Drugs Manual (1990) Chapter 5, Mutagenicity Study, Section 4: Chromosomal aberration test with mammalian cells in culture, pp. 50-55. Kung, A.L., S.W. Sherwood and R.T. Schimke (1990) Cell line-specific differences in the control of cell cycle progression in the absence of mitosis, Proc. Natl. Acad. Sci. (U.S.A.), 87, 9553-9557. Linnainmaa, K., and S. Wolff (1982) Sister chromatid e x -
16 change induced by short-lived monoadducts produced by the bifunctional agents mitomycin C and 8-methoxypsoralen, Environ. Mol. Mutagen., 4, 239-247. Loveday, K.S., M.H. Lugo, M.A. Resnick, B.E. Anderson and E. Zeiger (1989) Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro, II. Results with 20 chemicals, Environ. Mol. Mutagen., 13, 60-94. Margolin, B.H., B.J. Collings and J.M. Mason (1983) Statistical analysis and sample size determinations for mutagenicity experiments with binomial responses, Environ. Mutagen., 5, 705-716. Natarajan, A.T., A.D. Tates, P.P.W. van Buul, M. Meijers and N. de Vogel (1976) Cytogenetic effects of mutagens/ carcinogens after activation in a microsomal system in vitro, I. Induction of chromosome aberrations and sister chromatid exchanges by diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) in CHO cells in the presence of rat-liver microsomes, Mutation Res., 37, 83-90. Perry, P. and S. Wolff (1974) New giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-158. Preston, R.J., W. Au, M.A. Bender, J.G. Brewen, A.V. Carrano, J.A. Heddle, A.F. McFee, S. Wolff and J.S. Wassom (1981) Mammalian in vivo and in vitro cytogenetic assays:
A report of the U.S. EPA's Gene-Tox Program, Mutation Res., 87, 143-188. Savage, J.R.K. (1975) Classification and relationships of induced chromosomal structural changes, J. Med. Genet., 12, 103-122. Scott, D., B.J. Dean, N.D. Danford and D.J. Kirkland (1990) Metaphase chromosome aberration assays in vitro, in: D.J. Kirkland (Ed.), Basic Mutagenicity Tests: UKEMS Recommended Procedures, Cambridge University Press, Cambridge, pp. 62-86. Thust, R. (1982) Interindividual variation of carcinogen activation by human liver homogenates, A study using dimethylnitrosamine (DMN) and cyclophosphamide (CP) as precursor genotoxic agents and clastogenicity and induction of sister chromatid exchanges in Chinese hamster V79-E cells as endpoints, Arch. Geschwulstforsch., 52, 97-104. Thust, R., J. Mendel, H. Schwarz and R. Warzok (1980) Activity in clastogenicity and SCE assays and aberration kinetics in Chinese hamster V79-E cells, Mutation Res., 79, 239-248. Wolff, S. (1978) Chromosomal effects of mutagenic carcinogens and the nature of the lesions leading to sister chromatid exchange, in: H.J. Evans and D.C. Lloyd (Eds.), Mutagen-induced Chromosome Damage in Man, Yale University Press, New Haven, pp. 208-215.
3
MUTENV 08871
Evaluation of the need for a late harvest time in the assay for chromosome aberrations in Chinese hamster ovary cells Christian L. Bean and Sheila M. Galloway Merck Research Laboratories, W45-305, West Point, PA 19486, USA
(Received 19 October 1992) (Revision received 15 December 1992) (Accepted 29 January 1993)
Keywords: Chromosome aberrations; CHO, in vitro; Kinetics; Sampling time; Clastogens
Summary Harvest time is one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. In CHO cells we have found that for a variety of chemicals one harvest time near 20 h is optimal following a 3-h treatment (Bean et al., 1992). However, some guidelines for testing for regulatory purposes recommend an additional late harvest time 24 h after the first. We tested 10 diverse chemicals in CHO-WBL cells harvested 20-21 h and 42-44 h from the beginning of a 3-hr treatment. We added BrdUrd after treatment and recorded the total% of aberrant cells, and the proportions of aberrations (abs) in first (M1), second (M2) or later metaphases. The chemicals fell into 3 categories: ab yield greatly decreased at 44 h: benzo[a]pyrene, cadmium sulfate, chlorambucil, 2,6-diaminotoluene, 4-nitroquinoline N-oxide and mitomycin C (e.g., 37.0% cells with abs at 20 h and 1.0% at 44 h); ab yields similar at 20 and 44 h: 2-aminobiphenyl, eugenol and 8-hydroxyquinoline (e.g., 8.5% at 20 h and 7.0% at 44 h); and one, dimethylnitrosamine (DMN), which was detected at both times but gave a stronger response at 44 h than at 20 h (e.g., at 10 mM: 6.2% at 20 h and 25.0% at 44 h). This DMN effect was not seen in normal diploid human cells. For DMN the higher ab levels at 44 h than at 20 h were contributed by abs in M3 cells. Thus, while for some chemicals ab yields decrease with successive division, further increases can be seen in CHO in later metaphases, notably for DMN. Overall, however, after a 3-h pulse treatment of CHO cells a positive ab result could be obtained at the early harvest time (20 h) for all 10 chemicals.
The time at which metaphase cells are sampled after treatment is critical for detection of chromosomal aberrations (abs). Since abs induced by most chemicals are produced during
Correspondence: Christian L. Bean, Merck Research Laboratories, W45-305, West Point, PA 19486, USA.
DNA replication (Evans and Scott, 1969), the harvest time must be selected to allow cells to progress through S-phase after treatment. Based on studies of abs induced by ionizing radiation it was clear that abs should be scored in the first metaphase after they are formed to avoid loss by cell death during mitosis or conversion of the initial abs into complex derivatives during subsequent cell cycles (Carrano and Heddle, 1973; Das
and Sharma, 1987). Thus, a sampling time of no more than 1 cell-cycle length from the beginning of treatment has been used, e.g., about 10 h from the beginning of a 3-h pulse treatment in CHO ceils which have a cell-cycle length of 12-14 h (Galloway et al., 1985). Because cell-cycle delay often results from clastogen exposure, that protocol was modified to allow delayed cells time to progress to mitosis; sampling times of, e.g., 18-28 h have proved appropriate in CHO cells (e.g., Galloway et al., 1987; Loveday et al., 1989). There are ongoing international discussions of appropriate harvest times for standard testing protocols for in vitro chromosome ab assays. The UKEMS guidelines (Scott et al., 1990) recommend a first harvest at about 1.5 cell-cycle lengths from the beginning of treatment, with a second harvest 24 h later, in cases where negative or equivocal results are seen at the first sampling time. Japanese guidelines (1990) recommend both 24 and 48 h sampling times, but these harvests are done after continuous treatment for 24 or 48 h in contrast to our current protocol with a 3-h treatment. We reported previously that for a variety of chemicals a single harvest time about 17-21 h after the beginning of a 3-h pulse treatment was adequate for ab detection when doses were appropriately selected (Bean et al., 1992). In a study of V79 hamster ceils Thust et al. (1980) found maximal ab yields occurred at widely different times (e.g., 24-60 h) after a 1-h pulse treatment even among a group of closely related chemicals. Here we explore further the chromosome ab kinetics for 10 chemicals by harvesting at 20-21 and 42-44 h from the beginning of a 3-h treatment. Few chemicals other than some nitroso compounds in Chinese hamster V79-E cells (Thust et al., 1980; Thust, 1982) and nitrogen mustard and maleic hydrazide with plant ceils (Evans and Scott, 1964, 1969) have been investigated in this way. Some of the chemicals and concentrations in the present study give weak or equivocal responses. The chemicals vary widely in their modes of action and include alkylating and crosslinking agents, a metal salt and a catechol. Abs were recorded in cells labelled with 5bromodeoxyuridine to determine how many cycles each metaphase cell had undergone since treatment.
Materials and methods
Culture of CHO WBL cells CHO cells were maintained in McCoy's 5A medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 2 mM L-glutamine, 100 U / m l penicillin and 100 /zg/ml streptomycin (all Gibco). Cultures were incubated at 37°C in a humidified, 5% CO 2 atmosphere. Metabolic activation system Liver homogenate ($9 fraction) was prepared from phenobarbital//3-napthoflavone treated male Sprague-Dawley [CRCD Crl:CD(SD)BR] rats from Charles River Breeding Farms, Raleigh, NC. $9 was stored at -70° C to -80°C and thawed immediately before use. $9 was mixed with sodium NADP (Boehringer Mannheim), and trisodium isocitrate (Sigma) in serum-free medium immediately before use. The final concentrations in cultures were: $9, 15 /xl/ml; NADP, 0.8 m g/ m l (1.05 mM) and trisodium isocitrate, 1.5 mg/ml (5.8 mM). Test chemicals and solvents 2-Aminobiphenyl (2-ABP; Cas No. 90-41-5), benzo[a]pyrene (BP; CAS No. 50-32-8), cadmium sulfate (CdSO4; CAS No. 10124-36-4), chlorambucil (CAB; CAS No. 305-03-3), dimethylnitrosamine (DMN; CAS No. 62-75-9), dimethyl sulfoxide (DMSO), 8-hydroxyquinoline (8-HQ; CAS No. 148-24-3), Mitomycin C (MMC; CAS No. 50-07-7) and 4-nitroquinoline N-oxide (4NQO; CAS No. 56-57-5) were from Sigma, St. Louis, MO. 2,6-Diaminotoluene (2,6-DAT; CAS No. 823-40-5), and eugenol (EUG; CAS No. 9753-0) were from Aldrich. Deionized, distilled water (dH20) was purchased from Gibco. CdSO4, DMN and MMC were prepared in dH20.2-ABP, BP, 2,6-DAT, EUG and 8-HQ were prepared in DMSO. 2-ABP, BP, DMN, EUG and 8-HQ were tested in the presence of $9 metabolic activation. None of the chemicals changed the pH of the medium substantially as judged by the color of the indicator. Assessment of chromosome-aberration kinetics About 24 h before treatment, 1.2 x 106 cells were seeded in 10 ml medium in 75-cm 2 flasks
(Corning Glass Works, Corning, NY). Just before treatment, the medium was replaced. For tests without $9, 9.9 ml of complete medium was added. For tests with $9 mix, cultures were rinsed once with prewarmed (37°C) Dulbecco's phosphate-buffered saline without Ca z+ and Mg 2+ (DPBS, Gibco) and refed with 9.9 ml of serumfree medium containing the $9 metabolic activation system. Test compound (100 ttl) was added from concentrated stocks (100 × ), and cultures both with and without $9 mix were incubated for 3 h at 37°C, then washed twice with warmed DPBS and refed with 10 ml of prewarmed complete medium containing 10 /~M 5-bromo-2'-deoxyuridine (BrdUrd, Sigma). BrdUrd was present during the 3-h treatment with BP, but not in later experiments, during treatment with all other chemicals. Cultures were harvested at 20 and 44 h (or 21 and 42 h for benzo[a]pyrene) from the beginning of treatment, and 2.5-3 h after addition of 0.1 /zg/ml colcemid (Gibco). Cells were harvested by mitotic shake-off, or by trypsinization to allow cell counts (Coulter counter) for cytotoxicity estimation. Cells were treated with hypotonic KCI (75 raM) for 1-3 rain at room temperature, washed twice with fixative (methanol:glacial acetic acid, 3:1 v/v), dropped onto slides and air-dried. Slides were stained for sister chromatid differentiation by the fluorescence plus Giemsa technique (Perry and Wolff, 1974) as modified by Goto (1978).
Toxicity assesssment Toxicity was assessed by estimates of monolayer confluence and observations of abnormal cell morphology, or by Coulter cell counts of trypsinized cultures. Estimates of confluence were generally within 10% of Coulter cell counts when both measurements were made.
Dose selection The maximum concentrations to be tested were based on our previous experiments in CHO cells by the same protocol and generally did not exceed 50% toxicity, based on reductions of cell counts at 20 h compared with concurrent controls. Lower doses were also selected based on previous experiments and were chosen to yield
weak to moderate effects. The maximum concentration tested was less than 10 mM except for CAB, DMN and 2,6-DAT which were tested up to 20 mM, 18 mM and 20 mM, respectively, due to lack of cytotoxicity. The osmolality of the medium was not increased over control medium with solvent.
Metaphase classification Abs were scored in cells classified in the following categories: M1, even, dark staining on both chromatids. M1 + , 1 partial S phase in BrdUrd followed by 1 complete S phase: regions with sister-chromatid differentiation (SCD), and regions with both chromatids still dark. These cells are in their second metaphase since treatment. M2, 2 complete S phases in BrdUrd: full SCD. M2 + , 1 partial S phase in BrdUrd followed by 2 complete S phases: regions with SCD and regions with even, light staining on both chromatids. These cells are in their third metaphase since treatment. M3, 3 complete S phases in BrdUrd: about three-quarters of the chromatin appears evenly, light stained with the remainder dark.
Aberration scoring At 20 h, 200 cells per point (100 M1 and 100 M1 + , where possible) were scored for abs unless there were very high ab frequencies. At 44 h, we scored 100 or 200 consecutive cells for abs, and recorded the division each cell was in, e.g., M1, M2. We used metaphases with 19-23 chromosomes, the modal number being 21. All types of structural abs were recorded; chromatid and isochromatid gaps were recorded but not ineluded in the totals of abs. We defined a gap as an achromatic region equal to or smaller than the width of a chromatid in that cell, or a larger lesion with visible connecting material across the gap. We calculated both the percentage of aberrant cells (the number of cells with structural abs per 100 cells) and the frequency of abs (the total number of abs per 100 cells), since a cell may have more than one ab. Ceils with 10 or more abs were classed as severely damaged (SD) cells, and scored as one aberrant cell but as 10 abs.
Mitomycin C
Benzo(a) pyrene
~ 6 0 1~--]21 HR ~ 4 2 HR
~.70 E~20 HR ~ 4 4 HR
"
~ 5o ~ 4o
~3o l- 20
~ 20
,<10
~1o <
0
0.35/JM
0.50 ,uM
"
0
1.00 pM
20 pM
Dose
Cadmium
8
40/JM
Dose
2,6-Diaminotoluene
Sulfate
T E~]20 HR ~ 4 4 HRJ
~20 "o
"k
~15 -o
~-
-
.
1~20 HR [ ] 4 4 HR I
8
7 15
7 ~I0
_.m - -
.
E 5
<
<
°~ o
1.0/JM
~
1,5/JM
~
o
10 mM
Dose
4-Nitroquinoline
~30
14 mM
18 mM
Dose
4-Nitroquinoline N-oxide
N-oxide
~ 3 0 r~]20 HR [ ] 4 4 HR
~]20 HR [ ] 4 4 HR
.k
o 25
=o25
-o
~-- 2 0
~C2o
815
81s
=o)
c .~10
~r
~s 0
o~ o 0.25/JM
0.50/JM
1 O0 ,uM
Chlorambucil
o 25 "1o
.
.
.
.
.
.
.
.
.
J .
_ ,\\\ I\\\11 iX\\ll r\\\
....
8 15 clO
0
5 mM
10 mM Dose
1.00 uM
Dose
Dose
A 3 0 1%--]20 HR [ ] 4 4 HR
J
20 mM
2.50 ,uM
For selected points, the percentage of aberrant cells in treated cultures was compared to concurrent controls by the "normal test" of Margolin et al. (1983), a version of the Chi square test based on a standard normal approximation. The following abbreviations are used in the text and figures: TD, chromatid deletion, TE, chromatid exchange, CD, isochromatid (chromosome) deletion and CE, chromosome exchange. Results Controls The mean percentage of cells with abs for negative and solvent controls was 2.5% (range 0.5-6.0%, 11 determinations). Concurrent control percentages were subtracted from treated values for all data shown in the figures. Aberration yields at two harvest times The chemicals can be grouped into 3 categories with regard to ab responses at the two harvest times. Those in Group I (BP, CdSO4, CAB, 2,6-DAT, 4-NQO and MMC) had substantial numbers of abs at 20 h but few abs remained at 44 h (Fig. 1). The lower ab yield observed at 20 h for 40 ~M BP than for 20 ~ M BP may be the result of differing degrees of cell cycle delay at these 2 doses such that the maximum yield of aberrations at 40 tzM BP might not be reached by 20 h. For 3 clastogens, 2-ABP, EUG and 8-HQ (Group II) the ab increases at 20 h were similar to those at 44 h (Fig. 2a). Similar results were observed in a repeat experiment. One chemical, DMN (Group III), was positive both at 20 and at 44 h but gave a much stronger response at 44 h (Fig. 2b). Distribution o f aberrations over successive cell cycles Cell-cycle delay seen from % M1 and M1 + ceils at 20 h is shown in Table 1. Control cultures had substantial numbers of M1 + cells at 20 h,
i.e., 62-83%. For the Group I chemicals, abs were scored at doses that yielded greater than 60% survival (cell counts or monolayer confluence) compared with controls at 20 h. The distribution of abs across division stages was comparable at all dose levels, so the cells scored were pooled across dose levels for the following analyses. Most of the abs seen for Group I chemicals at 20 h were in M1 cells (Table 2). Where second metaphase (M1 + ) cells were seen at 20 h, few had abs. At 44 h, the few abs present were observed in all divisions, M1 through M3, though M3 cells generally had fewer abs than M2 cells. For Group II chemicals (similar yields at 20 and 44 h) abs at 20 h were predominantly in M1 cells; very few more advanced cells were seen (Tables 1 and 2), due to cell-cycle delay. Quite toxic doses were required to induce abs for these Group II chemicals, i.e., cell counts were reduced to about 50% of controls. At 44 h (Table 2) 20-50% of the few remaining M1 cells contained abs (e.g., 2-ABP 3 ab cells/14; EUG, 10 ab cells/21) in contrast to 6-14% of the M1 cells at 20 h. Thus, there was a tendency for the extremely delayed M1 cells found at 44 h to contain abs. For 2-ABP and EUG, a reduction in abs with successive divisions was seen (Table 2): overall percentages of aberrant cells were lower in second than in first division cells (e.g., EUG, 16.7% of 48 M2 cells el. 50% of 21 M1 cells) and lower still in third division cells (EUG, 7.8% of 231 M3 cells). These observations were confirmed in repeat experiments (data not shown). However, for 8-HQ the abs at 44 h were all in third division cells (8.2% of 195 M3 cells); few earlier division cells were observed (Table 2). DMN yielded abs at 20 h in both M1 (7.3% of 300) and M1 + (second division) cells (4.6% of 373, P = 0.03 cf. control of 1.3%). Most of the abs observed at 44 h were in third division cells (17.0% of 276 M3 cells) but a higher percentage of aberrant cells was found in the few second division cells observed (69.6% of 23 M2 cells).
Fig. 1. Group I chemicals. Chromosome aberration induction at 20 or 21 h and 42 or 44 h after a 3-h treatment in CHO cells. Control rates have been subtracted from data shown. 200 cells scored per point at 20 or 21 h except when rate was over 30%, 100 cells scored, or over 50%, 50 cells scored. 100 cells scored at 42 or 44 h. * indicates a statistically significant ( P < 0.05) increase over controls.
a 8-Hydroxyquinoline
Eugenol
~r
~ 1 0 ["~20 HR [ ] 4 4 HR
~2
8 o~
81
o
~ 4
c #_
<
< 1.2 mM
1.4 mM
1.6 mM
:iFJ 40 pM Dose
Dose
50 #M
2-Aminobiphenyl ~ 1 0 [X]20 HR ~ 4 4 HR
7
*
8
*
O.O mM
1.0 mM
Dose
b Dimethylnitrosamine ~ 4 0 [ ] 2 0 HR [ ] 4 4 HR /J
~ao 82o
.~I0 <
~ X
0
5 mM
10 mM
20 mM
Dose
Fig. 2. Chromosome aberration induction at 20 and 44 h after a 3-h t re a t me nt in C H O cells. Control rates have been subtracted from data shown. 200 or more cells scored per point at 20 h, 100 cells scored at 44 h. * indicates a statistically significant ( P < 0.05) increase over controls. (a) Group 1I chemicals. At 20 h for 1.6 mM E U G only 114 cells were available. (b) G roup I l l chemical.
Types of aberrations
primarily chromatid deletions (TD) and exchanges (TE; Fig. 3). Data shown are for the highest dose only since similar distributions CdSO4, were
At 20 h the abs scored for 5 of the 6 Group I chemicals, MMC, 2,6-DAT, CAB, 4-NQO and
TABLE 1 C E L L C Y C L E D E L A Y IN C H O C U L T U R E S Treatment
Dose
Percentage of cells per division 20h M1
44 h M1 +
M1
M2
M3
8
92
1 1
11 22 30
89 77 69
1
22 44
77 55
1
4 4 1
96 96 98
1
50 65
50 34
4 5
96 95 99
Group I Negative control a MMC
CdSO 4
2,6-DAT
BP CAB
4-NQO
17
83
0.35/~M 0.50/xM 1.00/xM
87 100 b 100
13
1.00 ~ M 1.50 ~ M
70 100
30
10.00 m M 14.00 m M 18.00 m M
19 43 40
81 57 60
20.00/~M 40.00/xM
100 100
5.00/x M 10.00/IM 20.00/xM
80 81 100
20 19
0.25/~M 0.50/~M 1.00/xM 2.50/xM
26 70 69 86
74 30 31 14
5
100 99 100 95
39
62
2
98
29
71
1 1
Group H Negative control a Solvent control
2
98
EUG
1.2 m M 1.4 m M 1.6 m M
100 100 100
8 2 11
13 12 23
79 86 66
2-ABP
0.9 m M 1.0 m M
100 100
9 5
34 59
57 36
40.0/~M 50.0/~M
99 95
1 5
2 3
98 97
20
80
2
98
40 30 44
60 70 56
1 5 17
99 95 82
a
8-HQ
Group III Solvent control a DMN
5 mM 10 m M 20 m M
a Representative control data since several experiments were done. b Predominantly M1 cells were observed.
1
10
were seen at all doses. In addition, 2,6-DAT induced severely damaged (SD) cells containing chromatid deletions and exchanges. The other Group I chemical, BP, yielded chromatid exchanges and isochromatid deletions (CD) at 21 h (Fig. 3). These abs were in M1 ceils since no more advanced ceils were available due to cellcycle delay or cell death. By 44 h, few abs remained for any of these 6 chemicals. For the Group II chemicals the percentages of cells with abs at 20 h were similar to those at 44 h (Fig. 2a). Chromatid deletions and exchanges and
isochromatid deletions were observed at 20 h for 2-ABP (data not shown since too few M3 cells were available) and for E U G (Fig. 4). At 44 h, for 2-ABP chromatid exchanges were seen in the few M1 ceils observed and isochromatid deletions were observed in M2 and M3 cells (data not shown since too few M3 cells were available, Table 2). For EUG, by 44 h abs were chromatid exchanges, isochromatid deletions and chromosome exchanges (CE) in M3 ceils. Some chromatid exchanges were found both in the few M1 cells, and in M3 cells (Table 2, Fig. 4). However,
TABLE 2 ANALYSIS OF ABERRANT CELLS BY METAPHASE CATEGORY Treatment
Dose
20 h
44 h
% ab
ab cells/cells scored
cells
M1
M1 +
% ab cells
ab cells/cells scored
M1
M2 a
M3 b
1/8
1/92
0/1 1/1
1/11 1/22 1/30
4/89 0/77 3/69
2/2
3/63
7/235
1/1
0/22 3/44
0/55
1/1
3/66
1/133 4/96 1/96
1/1
0/4 0/4 0/1
1/1
0/9
10/290
0/2
6/100 10/131
1/100 1/67
0/2
16/231
2/167
Group I Negative control c
MMC
0.35/zM 0.50/zM 1.00/zM
3.5
5/100
2/100
2.0
9.0 37.0 62.0
16/100 37/100 31/50
2/100 NA NA
5.0 1.0 5.0
84/250
2/100
5/100 36/200
2/100 NA
41/300
2/100
13/100 17/100 26/100
4/100 3/100 1/100
56/300
8/300
51/100 38/100
NA NA
Totals
CdSO4
1.00 /zM 1.50/zM
3.5 18.0
Totals
2,6-DAT
10.00 mM 14.00 mM 18.00 mM
8.5 10.0 13.5
Totals
BP d
20.00/zM 40.00/xM
51.0 38.0
5.00/~M 10.00/~M 20.00/zM
6.0 15.5 31.0
Totals
4-NQO
0.25 ~M 0.50 tzM 1.00/~M 1.00/~M 2.50/xM Totals
4.0 1.0 6.0
3.5 5.5
89/200
Totals
CAB
1.0 6.0
2.0 3.5 8.5 3.9 27.5
9/100 27/100 31/100
3/100 4/100 NA
67/300
7/200
2/100 5/100 15/100 10/100 57/200
2/100 2/100 2/100 0/156 6/29
89/600
12/485
5/98
0/4 2/5 0/1
7,/96 7/95 9/99
2/10
23/290
0/1
6/100 5/99
3/3
1/10 19/97
10/190 4/100
3/3
20/108
33/589
7,0 9.0 9.0
6.0 5.0 8.0 5.5 13.0
1/78
8/100
11 TABLE 2 (continued) Treatment
Dose
20 h % ab cells
44 h ab cells/cells scored M1 M1 +
% ab cells
ab cells/cells scored M1 M2 a
M3 b
Group H
Negative control c DMSO solvent control c EUG
1.0% 1.2 mM 1.4 mM 1.6 mM
2.5
5/100
0/100
2.0
0/2
2/98
0.5
0/100
1/100
1.0
0/2
1/98
7.0 7.0 14.0
14/200 14/200 16/114
Totals 2-ABP
0.9 mM 1.0 mM
40.0 ~M 50.0 tzM
10.0 6.0 20.0
44/414 8.0 6.0
Totals 8-HQ
NA NA NA
16/200 12/200
NA NA
1.0 7.0
28/400 6.4 8.1
Totals
13/200 17/200
0/2 0/10
30/400
0/12
5/8 2/2 3/11
1/13 1/12 6/23
4/79 3/86 11/66
10/21
8/48
18/231
0/9 3/5
0/34 3/59
1/57 1/36
3/14
3/93
2/93
0/2 0/3
5/98 11/9"
0/5
16/19
5.0 11.0
Group III
dH20 solvent control DMN
1%
2.3
4/100
2/159
3.5
0/5
5 mM 10 mM 20 mM
4.5 6.2 6.6
7/100 9/100 6/100
3/121 6/141 8/111
5.0 25.0 34.0
1/1
0/1 3/5 13/17
5/99 22/95 20/82
1/ 1
16/23
47/276
Totals
22/300
17/373
7/195
This category includes a small number of M1 + cells which although intermediate in staining, are in their second metaphase since treatment. b This category includes M2+ cells which although intermediate in staining, are in their third metaphase since treatment. c Representative control since several experiments were done. d Harvest times for BP were 21 and 42 h after dosing. NA Few or no M1 + cells available for analysis. The data in this table are absolute values and have not been corrected for control rates. The control range was 0.5-6.0%, mean 2.47%, 11 determinations. Where possible, 100 M1 and 100 M1 + cells were scored at 20 h, while at 44 h 100 or 200 consecutive cells were scored. a
at 1.6 m M in a r e p e a t e x p e r i m e n t with E U G , in which there was slightly g r e a t e r toxicity, the chrom a t i d exchanges were seen in M1 a n d M2 cells at 44 h. This illustrates how ab types can vary from o n e e x p e r i m e n t to the next d u e to slight c h a n g e s in cell-cycle kinetics, i.e., at 1.6 m M , 66% of the cells were M3 at 44 h in the first e x p e r i m e n t b u t only 30% were M3 in the second. Some of the
M1 cells r e c o r d e d at 44 h were severely d a m a g e d ( 6 / 2 1 M1 cells) b u t 11 o t h e r M1 cells f o u n d had n o visible d a m a g e ( T a b l e 2). I n contrast to the 44-h result, n o SD cells were observed at 20 h for E U G . T h u s with E U G , the heavily d a m a g e d (SD) cells were greatly delayed, a p p e a r i n g as M1 cells at 44 h. I n contrast, SD cells with 2 , 6 - D A T were seen at 20 h a n d were gone by 44 h. F o r 8-HQ,
12 M i t o m y c i n C - 1.0 p M
!I
r~]20HR- M1 ~ 4 4 H R - M 3
g 7O 6o
~4o
~30 ~'20 ~ u. 0
2 , 6 - D i a m i n o t o l u e n e - 18 m M
o
-10 g-
~s g SD
Data from 50 M1 cells and 69 M3 cells
Cadmium
~ 14
.
.
u_ 0
TD
Benzo(a)pyrene
S u l f a t e - 1.5 p M
.
.
TE CD CE Aberration Types
SD
Data from 100 M1 cells and 98 M3 cells
~50/~
[~20HR-M1 [ ] 4 4 H R - M 3 .
~44HR'M~
-~15
TE CD CE Aberration Types
TD
I~20HR'M1
8 20
.
- 20 pM
11~]21HR-M1 D 4 2 H R - M 3 J
-
__.=12
='40
v
8 ao
8 8
~6
4 u_ 0
4- 0 TD
TE CD CE Aberration Types
TD
SD
TE CD CE Aberration Types
......-~ SD
Data from 100 M1 cells and 100 M3 cells
Data from 200 M1 cells and 55 M3 cells
Chlorambucil - 20/JM
4-Nitroquinoline N-oxide - 2.50/3M /
= 35/'~
[ ] 20 HR-M1 [ ] 4 4 H R - M 3 1
3
5
/
~
-
M
3
J
~ 3o ,4, ~ 25 .I 8 20 4"
o-
~3o
\\
8 2o
~ 25
\\ \\ \\ \\
5
u_ 0
- • \\ \\ \\
\\
TD
TE CD CE Aberration Types
SD
TD
TE CD CE Aberration Types
SD
Data from 200 M1 cells and 100 M3 cells Data from 100 M1 cells and 99 M3 cells Fig. 3. Types of aberrations induced at 20 and 44 h after a 3-h treatment without $9, except BP, with $9. The following abbreviations are used: TD, chromatid deletion, TE, chromatid exchange, CD, isochromatid (chromosome) deletion, CE, chromosome exchange and SD, severely damaged cells, scored as one aberrant cell but as 10 abs. Control frequencies have been subtracted from each data point.
13
8 - H y d r o x y q u i n o l i n e - 50 pM
Eugenol - 1.6 mM ~15 ca
o°10
g
~. 5
== u. 0
TD
TE
CD
CE
SD
TD
Aberration Types
TE
CD
Aberration
Data from 114 M1 cells and 66 M3 cells
CE
SD
Types
Data from 200 M1 cells and 97 M3 cells
Dimethylnitrosamine - 20 mM
D
o=
o
L,
TD
TE
CD
CE
SD
Aberration Types Data from 100 M1 cells and 82 M3 cells
Fig. 4. Types of aberrations induced at 20 and 44 h after a 3-h treatment with S-9. The following abbreviations are used: TD, chromatid deletion, TE, chromatid exchange, CD, isochromatid (chromosome) deletion, CE, chromosome exchange and SD, severely damaged cells, scored as one aberrant cell but as 10 abs. Control frequencies have been subtracted from each data point.
the abs at 20 h were mainly chromatid deletions and exchanges in addition to some SD cells (Fig. 4). By 44 h, all abs were in M3 cells and these were primarily chromosome exchanges in addition to a few chromatid exchanges at 50 /xM. There were no SD cells at 44 h for 8-HQ. Thus, overall for the Group II chemicals there was loss of severely damaged cells through division, and derived types of abs were seen at later divisions (e.g., chromosome exchanges, CE). Most of the CE observed in later divisions were not associated with acentric fragments (AF) (e.g., 8HQ: 3 A F / 2 2 CE; EUG: 3 AF/21 CE). For DMN the ab increases at 20 h were low but significant, e.g., totals of 6.2% at 10 mM and 6.6% at 20 mM (P < 0.02 for both dose levels
compared with controls; Fig. 2b shows percentages with controls subtracted). The abs included chromatid deletions and exchanges and isochromatid deletions (Fig. 4). By 44 h the abs were mainly chromatid exchanges and isochromatid deletions. Some SD cells were scored at 44 h in M2 cells (not shown) but most of the abs at 44 h were in M3 cells. DMN is the only chemical for which SD cells appeared as cells beyond the first metaphase. Discussion
A general practice has been to score abs in the first metaphase following treatment, to detect primary abs (Evans and O'Riordan, 1975; Preston
14 et al., 1981). Abs can lead to cell death due to mechanical difficulties at anaphase a n d / o r loss of material in the daughter cells. Also, some abs may lead to derived-type abs such as dicentrics or rings (reviewed by Savage, 1975). Thus, scoring abs in cells other than first division metaphases could lead to erroneous estimates of clastogenic potential. Careful selection of harvest time is thus one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. In previous work with the C H O cell system, sampling times used to detect abs were 9.5-12 h and were extended to 18-28 h if there was evidence for substantial cell-cycle delay from the numbers of first- and second-division metaphases in preliminary sister-chromatid exchange assays (Galloway et al., 1985, 1987; Ivett et al., 1989; Loveday et al., 1989). More recently, we found that, following a 3-h treatment, one harvest time near 20 h was optimal for obtaining positive results in C H O cells for a range of chemicals (Bean et al., 1992). However, some published and proposed guidelines recommend an additional very late harvest time (24 h after the first) for chemicals that are equivocal or negative at a first sampling time about 1.5 cycle times from the beginning of treatment (Scott et al., 1990). H e r e we tested 10 diverse chemicals and found that after a 3-h pulse treatment of C H O cells a single harvest time about 20 h after the beginning of treatment was adequate for ab detection, although one of the 10 chemicals had a greater yield of abs at 44 than at 20 h. Three categories were observed: ab yields greatly decreased at 44 h: BP, CdSO4, CAB, 2,6-DAT, 4 - N Q O and MMC; ab increases detected equally at 20 and 44 h: 2-ABP, E U G and 8-HQ, and one chemical, DMN, that was detected at both times but gave a much stronger response at 44 h, mainly in M2 to M3 cells. For the G r o u p I chemicals few abs were detected at 44 h probably because most of the clastogenic damage was lethal. Such toxicity is consistent with results for two of these chemicals (MMC and CdSO 4) which showed little initial toxicity at 3 or 10 h after treatment but dramatic reductions in cell counts and A T P content by 24 h (Armstrong et al., 1992). Colony-forming ability
measured at 7 - 8 days was also reduced at doses that caused large numbers of abs i.e., 1.67 g M CdSO 4 and 1.0/xM MMC. In contrast, significant toxicity was observed immediately after the 3-h treatment with 2-ABP, E U G and 8-HQ at clastogenic dose levels (Armstrong et al., 1992). Since we added BrdUrd after treatment we were able to record the percentage of aberrant cells and the total number of abs in first (MI), second (M2) or later metaphases. The chromosome exchanges in later division cells seen with E U G and 8-HQ suggests conversion to derived types of abs with successive divisions. For D M N the higher ab levels at 44 h than at 20 h were contributed by M3 cells, and apparently were arising in the second and third cycles after treatment possibly as a result of long-lived lesions not expressed as chromosome abs through replication or repair in the first cell cycle. The presence of some chromatid exchanges in M3 cells with E U G suggests persistence of lesions induced by E U G also. Chromosomal effects of long-lived lesions have been observed for other chemicals in some cell types. In plant cells (Vicia faba root tips), Evans and Scott (1969) found that after treatment with nitrogen mustard in S phase the yield of abs was significantly higher at the second mitosis than at the first mitosis. Similar results had been reported for maleic hydrazide (Evans and Scott, 1964). In Chinese hamster cells, Ikushima and Wolff (1974) also found evidence for long-lived lesions that could induce chromosome abs during first, second or third replications following irradiation of G~ cells with X-rays or UV light. For a series of nitroso compounds (Thust et al., 1980) maximal ab increases occurred 24-60 h after l-h pulse treatments of Chinese hamster V79-E cells (doubling time of 12-14 h). For DMN, Thust (1982) observed a maximal yield of abs at 36 h after a 3-h treatment in V79-E cells. At 32 h there were chromatid exchanges and to a lesser extent chromatid breaks, but few chromosome exchanges. These observations by Thust (1982) are in agreement with our data for D M N in C H O cells. In both labs, while the maximum response was later, significant increases in abs were found as early as 20-24 h. In addition, Natarajan et al. (1976) found increases in abs with D M N in C H O
15
cells as early as 15 h after a one-hour treatment, but found much higher yields of abs at 24 h. Studying sister-chromatid exchanges (SCEs), Wolff (1978) found that when CHO cells synchronized in G 1 were exposed to ethyl methanesulfonate, methyl methanesulfonate, 4-nitroquinoline- 1-oxide or acetoxyacetylaminofluorene, long-lived lesions were produced that led to the formation of SCEs in the first, second and to a lesser degree third cell cycle after treatment. Similar results were reported by Linnainmaa and Wolff (1982) for angelicin, the monofunctional derivative of 8-methoxypsoralen, in combination with UVA light. We are examining further the occurrence of abs induced by some chemicals in first, second, third or later division cells. The persistence of lesions could be specific to the cell type, probably related to repair capacity and/or the stringency of control mechanisms allowing cell cycle progression to metaphase in the presence of genetic damage (Kung et al., 1990). Our preliminary results show that in contrast to the increase in abs with time in CHO ceils, the ab yield with DMN decreases with successive cell generations in freshly isolated human lymphocytes and in early passage normal human fibroblasts. When cells are harvested about 1.5 cell cycles after treatment the initial yield of abs in human cells is similar to or less than that in CHO cells but direct comparisons are difficult since optimal ab yields may vary due to slight differences in cell kinetics. Our preliminary work suggests an increase with time may be specific to chemical agents (certain alkylating agents) and cell types (may not occur in untransformed human cells). We are investigating the potential role of O6-alkylguanine-DNA alkyltransferase in the time course of ab production by alkylating agents. From the present study we conclude that after a 3-h treatment of CHO cells a 20-h harvest time effectively detected clastogenic chemicals and a later harvest time was not required even for chemicals that are difficult to detect as clastogens. Acknowledgements We thank Timothy Johnson for his excellent technical contributions.
References Armstrong, M.J., C.L. Bean and S.M. Galloway (1992) A quantitative assessment of the cytotoxicity associated with chromosomal aberration induction in Chinese hamster ovary cells, Mutation Res., 265, 45-60. Bean, C.L., M.J. Armstrong and S.M. Galloway (1992) Effect of sampling time on chromsome aberration yield for 7 chemicals in Chinese hamster ovary cells, Mutation Res., 265, 31-44. Carrano, A.V., and J.A. Heddle (1973) The fate of chromosome aberrations. J. Theoret. Biol., 38, 289-304. Das, B.C., and T. Sharma (1987) The fate of X-ray-induced chromosome aberrations in blood lymphocyte culture, Mutation Res., 176, 93-104. Evans, H.J., and M.L. O'Riordan (1975) Human peripheral blood for the analysis of chromosome aberrations in mutagen tests, Mutation Res., 31, 135-148. Evans, H.J. and D. Scott (1964) Influence of DNA synthesis on the production of chromatid aberrations by X-rays and maleic hydrazide in l/icia faba, Genetics, 49, 17-38. Evans, H.J., and D. Scott (1969) The induction of chromosome aberrations by nitrogen mustard and its dependence on DNA synthesis, Proc. Roy. Soc. B, 173, 491-512. Galloway, S.M., A.D. Bloom, M. Resnick, B.H. Margolin, F. Nakamura, P. Archer and E. Zeiger (1985) Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: comparison of results for 22 compounds in two laboratories. Environ. Mutagen., 7, 1-51. Galloway, S.M., M.J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A.D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J. Rimpo, B.H. Margolin, M.A. Resnick, B. Anderson and E. Zeiger (1987) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals. Environ. Mol. Mutagen., 10, Suppl. 10, 1-175. Goto, K., S. Maeda, Y. Kano and T. Sugiyama (1978) Factors involved in differential giemsa-staining of sister chromatids, Chromosoma, 66, 351-359. Ikushima, T. and S. Wolff (1974) UV-induced chromatid aberrations in cultured Chinese hamster cells after one, two, or three rounds of DNA replication, Mutation Res., 22, 193-201. Ivett, J.L., B.M. Brown, C. Rodgers, B.E. Anderson, M.A. Resnick and E. Zeiger (1989) Chromosome aberrations and sister chromatid exchange tests in Chinese hamster ovary cells in vitro, IV. Results with 15 chemicals, Environ. Mol. Mutagen., 14, 165-187. Japanese Guidelines for Toxicity Studies of Drugs Manual (1990) Chapter 5, Mutagenicity Study, Section 4: Chromosomal aberration test with mammalian cells in culture, pp. 50-55. Kung, A.L., S.W. Sherwood and R.T. Schimke (1990) Cell line-specific differences in the control of cell cycle progression in the absence of mitosis, Proc. Natl. Acad. Sci. (U.S.A.), 87, 9553-9557. Linnainmaa, K., and S. Wolff (1982) Sister chromatid e x -
16 change induced by short-lived monoadducts produced by the bifunctional agents mitomycin C and 8-methoxypsoralen, Environ. Mol. Mutagen., 4, 239-247. Loveday, K.S., M.H. Lugo, M.A. Resnick, B.E. Anderson and E. Zeiger (1989) Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro, II. Results with 20 chemicals, Environ. Mol. Mutagen., 13, 60-94. Margolin, B.H., B.J. Collings and J.M. Mason (1983) Statistical analysis and sample size determinations for mutagenicity experiments with binomial responses, Environ. Mutagen., 5, 705-716. Natarajan, A.T., A.D. Tates, P.P.W. van Buul, M. Meijers and N. de Vogel (1976) Cytogenetic effects of mutagens/ carcinogens after activation in a microsomal system in vitro, I. Induction of chromosome aberrations and sister chromatid exchanges by diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) in CHO cells in the presence of rat-liver microsomes, Mutation Res., 37, 83-90. Perry, P. and S. Wolff (1974) New giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-158. Preston, R.J., W. Au, M.A. Bender, J.G. Brewen, A.V. Carrano, J.A. Heddle, A.F. McFee, S. Wolff and J.S. Wassom (1981) Mammalian in vivo and in vitro cytogenetic assays:
A report of the U.S. EPA's Gene-Tox Program, Mutation Res., 87, 143-188. Savage, J.R.K. (1975) Classification and relationships of induced chromosomal structural changes, J. Med. Genet., 12, 103-122. Scott, D., B.J. Dean, N.D. Danford and D.J. Kirkland (1990) Metaphase chromosome aberration assays in vitro, in: D.J. Kirkland (Ed.), Basic Mutagenicity Tests: UKEMS Recommended Procedures, Cambridge University Press, Cambridge, pp. 62-86. Thust, R. (1982) Interindividual variation of carcinogen activation by human liver homogenates, A study using dimethylnitrosamine (DMN) and cyclophosphamide (CP) as precursor genotoxic agents and clastogenicity and induction of sister chromatid exchanges in Chinese hamster V79-E cells as endpoints, Arch. Geschwulstforsch., 52, 97-104. Thust, R., J. Mendel, H. Schwarz and R. Warzok (1980) Activity in clastogenicity and SCE assays and aberration kinetics in Chinese hamster V79-E cells, Mutation Res., 79, 239-248. Wolff, S. (1978) Chromosomal effects of mutagenic carcinogens and the nature of the lesions leading to sister chromatid exchange, in: H.J. Evans and D.C. Lloyd (Eds.), Mutagen-induced Chromosome Damage in Man, Yale University Press, New Haven, pp. 208-215.