Mutation
MUT
45
Research, 265 (1992) 45-60
0 1992 Elsevier
Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
05033
A quantitative assessment of the cytotoxicity associated with chromosomal aberration detection in Chinese hamster ovary cells Michael Merck
J. Armstrong, Sharp and Dohme
Christian
L. Bean and Sheila M. Galloway
Research Laboratories. (Received (Revision
I
Keywords: Chromosome-aberration
February
received
(Accepted
assays. in vitro: ATP: ACT:
WP45-305,
West Point,
PA ITI
(U.S.A.)
IYYI)
I I April
199 I)
I July 1991)
Mitotic
index; Colony forming; Sampling time
Summary
Regulatory guidelines suggest testing chemicals up to cytotoxic doses in chromosomal-aberration assays. To investigate the utility and limitations of various cytotoxicity indicators we used Chinese hamster ovary (CHO) cells to test 8 chemicals with differing ratios of cytotoxicity to clastogenicity. We measured immediate or delayed cell killing and growth inhibition (ATP levels, cell counts, colony-forming efficiency, CFE) and cell-cycle perturbations (mitotic index, MI; average generation time, AGT). Aberrations cabs) were scored 10 and 24 h from the beginning of the 3-h treatment. All 8 compounds induced abs at concentrations that reduced cell growth at 24 h by 50% or less. Concentrations of each chemical which induced at least 15% cells with abs, gave little loss of CFE (O-20%) for mitomycin C, adriamycin, cadmium sulfate and 2,6-diaminotoluene in contrast to the marked loss of CFE (70-80%) for eugenol (EUG), 2-aminobiphenyl and 8-hydroxyquinoline (8-HQ). 2,4-Diaminotoluene (2,4-DAT) was intermediate. Higher aberration yields were found at 24 h than at 10 h, even when minimal cell-cycle delay was detected by AGT estimates from BrdUrd-labeled cells. Cells with multiple abs were seen at 24 but not at 10 h, and often confirmed clastogenicity when there was only a weak increase in the percentage of cells with aberrations. Total ATP per culture did not always correlate with cell number, especially at later times after treatment. This is likely due to metabolic perturbations or altered cell biomass that are known to affect cell ATP content. MI suppression often did not correlate with AGT, e.g., only small increases in AGT were seen for 8-HQ, 2,4-DAT and EUG despite severe mitotic suppression at 10 h. By 24 h the MI for all chemicals had recovered, sometimes exceeding control levels. Marked mitotic accumulation was seen at 10 h for 2,4-DAT, indicating cell synchrony. Thus, the MI has limited value for dose selection. In conclusion, even weakly active chemicals were detected at a single time without exceeding a 50% growth reduction at 24 h.
Correspondence: Dohme
Research
19486 (U.S.A.).
Dr.
Sheila
M. Galloway,
Laboratories,
WP45-305,
Merck West
Sharp and Point,
PA
The chromosomal aberration assay in Chinese hamster cells is often applied as an indicator of in vitro genotoxicity, but quantitative assessment of
46 the toxicity associated with aberration (ab) induction is rarely done. Cytotoxicity data are needed in chromosome-aberration studies for dose selection, and are especially important when the results are used in risk assessment of compounds to which humans may be exposed. Cytotoxicity can be evaluated in vitro by methods such as measurement of ATP (Kemp, 1988), mitochondrial activity (Mosmann, 1983), inhibition of cell-cycle progression or loss of proliferative capacity. Comparative studies frequently have shown cases of poor agreement between measurements of metabolic dysfunction and of cell survival as measured by CFE (Bhuyan et aI., 1976; Weisenthal et al., 1983) and some claim that the only relevant indicator of toxicity in proliferating populations is reproductive death (Roper and Drewinko, 1976). Thus, it is important to know which toxicity indicators are sensitive and widely applicable to the variety of compounds routinely tested in the in vitro chromosome-aberration test. Consideration of cell-cycle delay is important in test design since the ab yield is highly dependent upon the sampling time (Ceccherini et al., 1988; Nowak, 1990; Bean et al., 1992). Since abs can be lost during mitosis it is important to choose the interval between treatment and fixation so that the cells collected for analysis are in their first metaphase (Carrano, 1973). However, the sampling time must also accommodate any treatment induced delay in cell progression, to allow damaged cells to reach their first metaphase. Indicators of perturbations in the cell cycle are thus useful not only as indices of toxicity but in selection of optimal harvest time. Here we measured growth suppression, immediate or delayed cell killing and perturbations in the cell cycle as assessments of the toxicity profiles of 8 clastogens of differing potencies, and to demonstrate the utility and limitations of the various cytotoxicity indicators in the CHO system. Delayed or reproductive death (colony-forming efficiency, CFE) was determined by two methods where cells were treated at high or low density. Growth suppression (10 and 24-29 h) and immediate cell killing following the 3-h treatment was assessed using dye exclusion. Cellular adenosine trimhn~nhate [ATP) level~ were evaluated at 3_ Ill
and 24 (or 29) h as an alternative rapid assessment of cell viability (Kangas et al., 1984). Cellcycle delay was estimated by average generation time (AGT) in 5'-bromodeoxyuridine labelled cells (Ivett and Tice, 1982). We have used A G T in the past (Galloway et al., 1987) to determine whether a later harvest time (20-24 h) was necessary to detect aberrations, as a supplement to the standard 10-12 h harvest designed to obtain first-metaphase cells. This was re-evaluated here, and we assessed the use of the mitotic index (MI) in selection of harvest time and test concentrations. To assess the influence of sampling time on ab yield, cells were harvested for ab analysis 10 and 24 h from the beginning of the 3-h treatment. Material and methods
Cells. CHO cells, clone WBL, were used at no more than 15 passages since cloning, and were cultured in McCoy's 5A medium with 10% fetal calf serum, 2 mM c-glutamine, 100 units/ml penicillin and 100/.~g/ml streptomycin (complete McCoy's; all Gibco) in a humidified atmosphere of 5% CO 2 in air, at 37 ° C. Cells were plated the day before treatment at 1.0-1.2 million ceils/10 ml/75-cm 2 flask. Chemicals. Cadmium sulfate (CdSO4: CAS No. 10124-36-4), 2-aminobiphenyl (2-ABP: CAS No. 90-41-5), mitomycin C (MMC: CAS No. 5007-7), adriamycin (ADR: CAS No. 25316-40-9), and 8-hydroxyquinoline (8-HQ: CAS No. 148-243) were obtained from Sigma. 2,4-Diaminotoluene (2,4-DAT: CAS No. 95-80-7), 2,6-diaminotoluene (2,6-DAT: CAS No. 823-40-5) and eugenol (EUG: CAS No. 97-53-0) were from Aldrich. Treatment. Exponentially growing cells received 3-h treatments with chemical delivered to fresh medium at 1% v / v from stocks prepared at the time of treatment in either DMSO (2-ABP, EUG, 2,4-DAT, 2,6-DAT, 8-HQ) or H 2 0 (ADR, MMC). CdSO 4 was dissolved directly into medium containing 10% fetal calf serum. Based on reported results for in vitro aberration induction, treatments were done either in the presence (2ABP_ EIJG. R-He) or absence (ADR. MMC.
47
2,4-DAT, 2,6-DAT, CdSO 4) of $9 activation. The activation medium was serum-free McCoy's 5A medium and 0.8 m g / m l N A D P (1.0 mM /3-nicot i n a m i d e - a d e n o s i n e dinucleotide p h o s p h a t e , sodium salt; Boehringer Mannheim), 1.5 m g / m l trisodium isocitrate (5.8 mM; Sigma) and 15 / , I / m l rat liver $9. The $9 was prepared from male Sprague-Dawley rats induced with phenobarbital and /3-naphthoflavone, and the protein concentration of the homogenate was about 40 m g / m l , so that the final concentration was ().6 m g / m l protein. Treatment without $9 was in complete McCoy's 5A medium. All treatments were terminated after 3 h by washing twice with prewarmed Dulbecco's phosphate-buffered saline (Gibco) and all endpoints were assessed in the same experiment using replicate cultures. Cell counts and ATP measurements at 3 h (and 10 h for CdSO 4) were done in a separate set of experiments.
Total cell counts. At 3, 10 and 24 or 29 h after the beginning of treatment, all cells were collected by trypsinization and 1-2 aliquots were diluted in 0.4% trypan blue (Gibco) for hemacytometer counts. From each aliquot 2 samples of 100-300 cells were counted for each treatment and the mean of the viable cell counts from thc 2 - 4 samples was expressed as a percentage of controls. Total culture A TP.
Total ATP was determined at 3, I0 and 24 or 29 h using the luciferin-luciferase bioluminescent reaction (St. John, 1970; Waters et al., 1975) measured in an LKB Luminometer (Model 125(I). Aliquots (0.1 ml) of cell suspensions were extracted with (1.9 ml of cold D M S O in M O P ' s buffer (90% v/v), vortexed and diluted to 6 ml with cold MOP's buffer (0.01 M, pH 7.5; Sigma). The extracts were stable for at least 24 h when stored at 4 ° C and DMSO at 12% v / v did not alter A T P measurements. For each treatment four 25-/,1 aliquots of the extracts were assayed for total A T P using 100 /,1 Lumit PM per sample (lyophilized luciferin-luciferase reconstituted with 0.025 M HEPES, pH 7.75, LKB) and the resulting bioluminescence was quantitated by computerized integration of the light reaction curve. The output was recorded as luminescence units and was proportional to the
ATP concentration of the sample. Results for the drug-treated cultures were calculated as total ATP per culture and expressed as a percentage of combined medium a n d / o r solvent controls. Variation between aliquots was typically less than 5% of the mean. For selected treatments, total ATP was divided by viable cell number to give the average ATP per cell.
Colony forming efficieno,. Two methods were used to determine colony-forming efficiency. In the 'replate' method, cells were washed at the end of the 3-h treatment, trypsinized, counted (Coulter counts) and plated at 100 cells/60-mm dish. In the 'in situ' method, cultures were seeded at 100 cells/60-mm dish, and 3 h later were treated, then washed after a further 3 h. All dishes (5 per treatment) from both methods were incubated in complete McCoy's 5A medium for 7 - 8 days, then colonies were stained with 0.4% methylene blue and counted. Results were expressed as a percentage of controls and the concentration which gave 50% relative survival (ECs0) was estimated from the d o s e - r e s p o n s e curve.
Acerage generation time estimates'. After the 3-h chemical treatment, cells were washed and allowed to recover for 26 h in complete McCoy's 5A medium supplemented with 5-bromodeoxyuridine at 10 /zM (BrdUrd; Sigma). About 3 h before harvest, colcemid (1(1 /xg/ml; G i b c o ) w a s added and accumulated mitotic cells were later collected by shakeoff, treated with hypotonic KCI (0.075 M) and fixed in 3 : 1 methanol : glacial acetic acid. Air-dried chromosome preparations were stained by the fluorescence-plus-Giemsa technique of Perry and Wolff (1974) modified by Goto et al. (1978). To estimate the average generation time (AGT), a replicative index (RI) based on 200 cells was calculated from the proportions of cells that had completed 1 (M1), 2 (M2) or between 1 and 2 (M1 + ) cycles in BrdUrd, RI = (M1 × 1 ) + ( M I + × 1 . 5 ) + ( M 2 × 2 ) . A G T (h) was calculated as the number of h in BrdUrd divided by the RI, and represents a rough estimate of the cell cycle time since all cells that had completed between 1 and 2 cycles were designated M1 + and treated as 1.5 cell cycles in the calculations.
48 TABLE 1 COMPARISON OF IN V I T R O M E T H O D S TO M E A S U R E T O X I C I T Y Treatments " dose
3hb
3hc
10 h
Counts
ATP
Counts
Counts
ADR (~M)
0.10 0.50 0.75 1.00
95 106 93 102
93 101 93 102
102 100 103 98
2-ABP (mM)
0.7 0.8 0.9 1.0 1.1 1.2
92 92 93 73 76 63
72 69 69 65 55 45
CdSO 4 (~M)
0.33 1.67 3.33
99 90 88
2,4-DAT (mM)
2 4 6 8
2,6-DAT (mM)
8 10 14 18
EUG (mM)
8-HQ (~M)
MMC (#M)
0.4 0.8 1.2 1.6 2.0 30 40 50 60 70 0.5 1.0 2.0 4.0 8.0
10 h
24-29 h
ATP
Counts
24-29 h ATP
MId
MI J
88 71 71 68
100 102 98 91
91 66 55 38
101 91 91 87
91 25 8 1
117 114 78 89
104 140 156 177
110 47 4 1
ND ND ND ND ND ND
96 77 84 66 59 8
94 92 84 76 55 9
94 79 57 57 28 8
82 75 72 61 33 8
97 57 64 49 4 3
97 88 67 69 63 14
106 112 133 151 183 188
72 64 83 58 23 2
102 85 77
ND ND ND
87 77 72
102 93 71
98 49 22
97 60 35
97 2 2
144 94 27
98 160 173
96 56 12
94 91 103 83
94 92 96 74
90 91 91 94
92 80 88 101
97 93 95 98
88 60 65 51
104 95 92 99
153 74 30 16
143 151 152 110
102 102 108 118
110 71 80 59
101 98 98 97
124 123 108 113
98 88 96 89
83 77 79 88
116 112 102 104
77 82 79 67
96 93 96 89
125 143 117 56
107 146 144 147
102 103 107 109
105 117 104 81
90 83 92 59 3
99 98 89 42 3
80 95 94 ,80 43
83 73 75 64 5
88 80 78 65 5
61 66 66 37 3
85 83 84 57 4
65 79 76 19 0
106 130 131 108 0
106 112 111 128 ND
69 114 76 59 0
99 46 22 13 6
100 45 24 14 5
94 97 84 73 62
89 71 59 26 18
94 78 61 30 17
96 48 32 15 9
102 77 53 23 15
100 1 3 1 1
92 42 21 19 13
101 114 110 107 107
65 25 18 19 5
109 104 96 105 ND
113 112 106 119 ND
74 83 78 73 67
99 89 99 97 86
105 111 110 100 107
96 69 57 67 51
94 96 101 90 90
34 33 26 25 11
100 153 95 61 25
122 176 191 > 194 > 194
89 48 10 2 0
AGT e
CFE f
Values are percentages of solvent, or combined solvent and negative controls. Viable cell number was assessed using trypan dye exclusion, except C3-h counts. a 3-h treatment. b These 3-h data for counts and ATP are from a separate experiment. All other data are from cultures treated concurrently. c Coulter cell counts. d Mitotic index. e Cell-cycle inhibition: A G T as % increase over control, control AGT ranged from 12.9 to 14.8 h. f Colony-forming efficiency: in-situ (2-ABP, CdSO 4) or replate data. ND, not determined concurrently with the other endpoints.
49
Mitotic index. At 10 and 24 or 29 h after the beginning of treatment all cells from colcemidtreated flasks were collected by trypsin harvest, fixed and stained with Giemsa. The mitotic index (MI) was the percentage of metaphase figures in 1000 cells. Chromosomal-aberration analysis. The A G T for C H O cells is 13-14 h. Colcemid arrested mitotic cells were harvested 10 and 24 h after the beginning of treatment. These cultures did not contain BrdUrd. Air-dried chromosome preparations were stained with Giemsa and 100-200 cells per treatment were scored for abs by one observer. Fewer than 200 cells were scored per treatment when ab frequencies were high or few cells were available for analysis due to mitotic suppression. Gaps, polyploid and endoreduplicated cells, and pulverized chromosomes were noted but not included in the ab totals. We classified as 'pulverized' chromatin that resembled prematurely condensed chromosomes from S-phase cells (Gollin et al., 1984). A cell with 10 or more abs was categorized as severely damaged and scored as 1 aberrant cell, but as 10 abs when calculating the total number of abs. Gaps were defined as achromatic lesions equal to or smaller than the width of the chromatid, and larger lesions with visible connecting material across the gap. Results are shown as the percentage of cells with abs (aberrant cells) and frequencies of abs per 100 cells. Statistical analysis of the percentages of cells with abs in treated cells compared with concurrent controls was by the 'normal test' of Margolin et al. (1983), a chi-squared test based on a standard normal approximation. Results
Details of toxicity and ab results are discussed for each compound below. Representative data comparing results of the toxicity endpoints are compiled in Table 1.
Adriamycin (Fig. 1) No toxicity as measured by cell counts was seen immediately after treatment for any dose between 0.1 and 2.0 /zM. A slight reduction in
24-
A
- ~ - t0HrMI
20 - % %
gv
12
28
-O-~ AGT24 Hr MI
24 if) -..q
d
20 "~
O c
8
-16 v
40100-
12 B
1
_~ -~ 80-
114
°t
I
E¢~ 60- ~ t~ .~ 40-
10 Hr 24Hr !
O-
120-
-
-
C
100 80
o~ 6o "6 ~ 40 20-
-~~ t0 HrCounts 24 Hr Counts ~
\
-
CFE
~m 0
II
0
~
~
0.10 0.25
~
0.50 ADR (pM)
~
0.75
1.00
Fig. 1. Cytotoxicity and chromosome aberration induction by adriamycin after 3-h treatment without $9: (A) MI at 10 and 24 h and AGT; (B) structural aberrations at 10 and 24 h, shown as % aberrant cells (% ab cells). The numbers above bars are frequencies of aberrations per 100 cells (shown when markedly different from % ab cells). % ab cells at all doses were significantly greater than combined controls, p < 0.05; (C) cell counts at 3, 10 and 24 h and CFE (replate).
cell number was observed at 10 h with doses between 0.1 and 1.0/zM while the CFE suppression was more than 50% at 0.5 tzM and above. Aberrations were induced by A D R at very low concentrations (17.5% aberrant cells at 0.1 /zM) in the absence of any acute or delayed toxicity. At 1.0 /zM, the few mitotic cells seen at 10 h had severe chromosome damage. More abs were seen at 10 than at 24 h at all concentrations of A D R . At low concentrations there was a lack of mitotic suppression or of change in A G T and as might be expected there was a higher ab yield at the earlier
50 h a r v e s t time. B u t at h i g h e r c o n c e n t r a t i o n s s u c h as 0 . 5 / z M , A D R was u n u s u a l in t h a t e v e n w h e n t h e M I a n d A G T i n d i c a t e d cell-cycle d e l a y t h e yield o f abs was h i g h e r at 10 t h a n 24 h a l t h o u g h it m i g h t be e x p e c t e d t h a t d e l a y e d cells w o u l d app e a r at 24 h. T h e d e c r e a s e in abs w i t h t i m e c o u l d be d u e to d e a t h o r a r r e s t o f cells w i t h abs, b u t if so, o n l y a small p r o p o r t i o n o f t h e cell p o p u l a t i o n is a f f e c t e d since t h e i n c r e a s e in a b s o l u t e cell n u m b e r f r o m 10 to 24 h s h o w e d t h a t at 0.5 # M cells w e r e g r o w i n g as fast as c o n t r o l s ( d a t a n o t
20-
:s
-
24
12 - 20
N
a
"7O E
-16 ~
4 0 25
> O
Q) ..D
< o4
10
/,'
I
I
I
I
12
~
27!
B
17 ~
i
24Hr
16~ 12
B
444
~o 8O 60
10 Hr 24 Hr
40
118
O4 2O 0 100 80-
"5 O4 40-
10
|
1
~
-~- 10HrCounts - I - 29 HrCounts - ¢ ~ ~ ~
0 ----F-/ 0 0.7
~
~
0.8 0.9 1.0 2-ABP (raM)
~. \ ~ \ 1 ~ 1.1
3 Hr Counts
~ -0-
0 Hr C
o
u
n
29 Hr C
o
u
n
I I
1.2
Fig. 2. Cytotoxicity and chromosome aberration induction by 2-aminobiphenyl after 3-h treatment with $9: (A) MI at 10 and 29 h and AGT; (B) structural aberrations at 10 and 24 h, shown as % aberrant cells (% ab cells). The numbers above bars are frequencies of aberrations per 100 cells (shown when markedly different from % ab cells). %. ab cells at 1.1 and 1.2 mM at 24 h were significantly greater than DMSO control, P < 0.05; (C) cell counts at 3, 10 and 29 h and CFE (in situ). 3-h counts were from a separate experiment.
t
s t
~ s
~
CFE
~
0 0.3
60 O4 20
o E
~1
10 Hr
0 - ~ ¢ ~---t----100 8O C ~
,.6 40
8-
20-
5
o
--4
20 "~
60I
20 0 15 Erd
©
0 100-
43
MUT
45
Research, 265 (1992) 45-60
0 1992 Elsevier
Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
05033
A quantitative assessment of the cytotoxicity associated with chromosomal aberration detection in Chinese hamster ovary cells Michael Merck
J. Armstrong, Sharp and Dohme
Christian
L. Bean and Sheila M. Galloway
Research Laboratories. (Received (Revision
I
Keywords: Chromosome-aberration
February
received
(Accepted
assays. in vitro: ATP: ACT:
WP45-305,
West Point,
PA ITI
(U.S.A.)
IYYI)
I I April
199 I)
I July 1991)
Mitotic
index; Colony forming; Sampling time
Summary
Regulatory guidelines suggest testing chemicals up to cytotoxic doses in chromosomal-aberration assays. To investigate the utility and limitations of various cytotoxicity indicators we used Chinese hamster ovary (CHO) cells to test 8 chemicals with differing ratios of cytotoxicity to clastogenicity. We measured immediate or delayed cell killing and growth inhibition (ATP levels, cell counts, colony-forming efficiency, CFE) and cell-cycle perturbations (mitotic index, MI; average generation time, AGT). Aberrations cabs) were scored 10 and 24 h from the beginning of the 3-h treatment. All 8 compounds induced abs at concentrations that reduced cell growth at 24 h by 50% or less. Concentrations of each chemical which induced at least 15% cells with abs, gave little loss of CFE (O-20%) for mitomycin C, adriamycin, cadmium sulfate and 2,6-diaminotoluene in contrast to the marked loss of CFE (70-80%) for eugenol (EUG), 2-aminobiphenyl and 8-hydroxyquinoline (8-HQ). 2,4-Diaminotoluene (2,4-DAT) was intermediate. Higher aberration yields were found at 24 h than at 10 h, even when minimal cell-cycle delay was detected by AGT estimates from BrdUrd-labeled cells. Cells with multiple abs were seen at 24 but not at 10 h, and often confirmed clastogenicity when there was only a weak increase in the percentage of cells with aberrations. Total ATP per culture did not always correlate with cell number, especially at later times after treatment. This is likely due to metabolic perturbations or altered cell biomass that are known to affect cell ATP content. MI suppression often did not correlate with AGT, e.g., only small increases in AGT were seen for 8-HQ, 2,4-DAT and EUG despite severe mitotic suppression at 10 h. By 24 h the MI for all chemicals had recovered, sometimes exceeding control levels. Marked mitotic accumulation was seen at 10 h for 2,4-DAT, indicating cell synchrony. Thus, the MI has limited value for dose selection. In conclusion, even weakly active chemicals were detected at a single time without exceeding a 50% growth reduction at 24 h.
Correspondence: Dohme
Research
19486 (U.S.A.).
Dr.
Sheila
M. Galloway,
Laboratories,
WP45-305,
Merck West
Sharp and Point,
PA
The chromosomal aberration assay in Chinese hamster cells is often applied as an indicator of in vitro genotoxicity, but quantitative assessment of
46 the toxicity associated with aberration (ab) induction is rarely done. Cytotoxicity data are needed in chromosome-aberration studies for dose selection, and are especially important when the results are used in risk assessment of compounds to which humans may be exposed. Cytotoxicity can be evaluated in vitro by methods such as measurement of ATP (Kemp, 1988), mitochondrial activity (Mosmann, 1983), inhibition of cell-cycle progression or loss of proliferative capacity. Comparative studies frequently have shown cases of poor agreement between measurements of metabolic dysfunction and of cell survival as measured by CFE (Bhuyan et aI., 1976; Weisenthal et al., 1983) and some claim that the only relevant indicator of toxicity in proliferating populations is reproductive death (Roper and Drewinko, 1976). Thus, it is important to know which toxicity indicators are sensitive and widely applicable to the variety of compounds routinely tested in the in vitro chromosome-aberration test. Consideration of cell-cycle delay is important in test design since the ab yield is highly dependent upon the sampling time (Ceccherini et al., 1988; Nowak, 1990; Bean et al., 1992). Since abs can be lost during mitosis it is important to choose the interval between treatment and fixation so that the cells collected for analysis are in their first metaphase (Carrano, 1973). However, the sampling time must also accommodate any treatment induced delay in cell progression, to allow damaged cells to reach their first metaphase. Indicators of perturbations in the cell cycle are thus useful not only as indices of toxicity but in selection of optimal harvest time. Here we measured growth suppression, immediate or delayed cell killing and perturbations in the cell cycle as assessments of the toxicity profiles of 8 clastogens of differing potencies, and to demonstrate the utility and limitations of the various cytotoxicity indicators in the CHO system. Delayed or reproductive death (colony-forming efficiency, CFE) was determined by two methods where cells were treated at high or low density. Growth suppression (10 and 24-29 h) and immediate cell killing following the 3-h treatment was assessed using dye exclusion. Cellular adenosine trimhn~nhate [ATP) level~ were evaluated at 3_ Ill
and 24 (or 29) h as an alternative rapid assessment of cell viability (Kangas et al., 1984). Cellcycle delay was estimated by average generation time (AGT) in 5'-bromodeoxyuridine labelled cells (Ivett and Tice, 1982). We have used A G T in the past (Galloway et al., 1987) to determine whether a later harvest time (20-24 h) was necessary to detect aberrations, as a supplement to the standard 10-12 h harvest designed to obtain first-metaphase cells. This was re-evaluated here, and we assessed the use of the mitotic index (MI) in selection of harvest time and test concentrations. To assess the influence of sampling time on ab yield, cells were harvested for ab analysis 10 and 24 h from the beginning of the 3-h treatment. Material and methods
Cells. CHO cells, clone WBL, were used at no more than 15 passages since cloning, and were cultured in McCoy's 5A medium with 10% fetal calf serum, 2 mM c-glutamine, 100 units/ml penicillin and 100/.~g/ml streptomycin (complete McCoy's; all Gibco) in a humidified atmosphere of 5% CO 2 in air, at 37 ° C. Cells were plated the day before treatment at 1.0-1.2 million ceils/10 ml/75-cm 2 flask. Chemicals. Cadmium sulfate (CdSO4: CAS No. 10124-36-4), 2-aminobiphenyl (2-ABP: CAS No. 90-41-5), mitomycin C (MMC: CAS No. 5007-7), adriamycin (ADR: CAS No. 25316-40-9), and 8-hydroxyquinoline (8-HQ: CAS No. 148-243) were obtained from Sigma. 2,4-Diaminotoluene (2,4-DAT: CAS No. 95-80-7), 2,6-diaminotoluene (2,6-DAT: CAS No. 823-40-5) and eugenol (EUG: CAS No. 97-53-0) were from Aldrich. Treatment. Exponentially growing cells received 3-h treatments with chemical delivered to fresh medium at 1% v / v from stocks prepared at the time of treatment in either DMSO (2-ABP, EUG, 2,4-DAT, 2,6-DAT, 8-HQ) or H 2 0 (ADR, MMC). CdSO 4 was dissolved directly into medium containing 10% fetal calf serum. Based on reported results for in vitro aberration induction, treatments were done either in the presence (2ABP_ EIJG. R-He) or absence (ADR. MMC.
47
2,4-DAT, 2,6-DAT, CdSO 4) of $9 activation. The activation medium was serum-free McCoy's 5A medium and 0.8 m g / m l N A D P (1.0 mM /3-nicot i n a m i d e - a d e n o s i n e dinucleotide p h o s p h a t e , sodium salt; Boehringer Mannheim), 1.5 m g / m l trisodium isocitrate (5.8 mM; Sigma) and 15 / , I / m l rat liver $9. The $9 was prepared from male Sprague-Dawley rats induced with phenobarbital and /3-naphthoflavone, and the protein concentration of the homogenate was about 40 m g / m l , so that the final concentration was ().6 m g / m l protein. Treatment without $9 was in complete McCoy's 5A medium. All treatments were terminated after 3 h by washing twice with prewarmed Dulbecco's phosphate-buffered saline (Gibco) and all endpoints were assessed in the same experiment using replicate cultures. Cell counts and ATP measurements at 3 h (and 10 h for CdSO 4) were done in a separate set of experiments.
Total cell counts. At 3, 10 and 24 or 29 h after the beginning of treatment, all cells were collected by trypsinization and 1-2 aliquots were diluted in 0.4% trypan blue (Gibco) for hemacytometer counts. From each aliquot 2 samples of 100-300 cells were counted for each treatment and the mean of the viable cell counts from thc 2 - 4 samples was expressed as a percentage of controls. Total culture A TP.
Total ATP was determined at 3, I0 and 24 or 29 h using the luciferin-luciferase bioluminescent reaction (St. John, 1970; Waters et al., 1975) measured in an LKB Luminometer (Model 125(I). Aliquots (0.1 ml) of cell suspensions were extracted with (1.9 ml of cold D M S O in M O P ' s buffer (90% v/v), vortexed and diluted to 6 ml with cold MOP's buffer (0.01 M, pH 7.5; Sigma). The extracts were stable for at least 24 h when stored at 4 ° C and DMSO at 12% v / v did not alter A T P measurements. For each treatment four 25-/,1 aliquots of the extracts were assayed for total A T P using 100 /,1 Lumit PM per sample (lyophilized luciferin-luciferase reconstituted with 0.025 M HEPES, pH 7.75, LKB) and the resulting bioluminescence was quantitated by computerized integration of the light reaction curve. The output was recorded as luminescence units and was proportional to the
ATP concentration of the sample. Results for the drug-treated cultures were calculated as total ATP per culture and expressed as a percentage of combined medium a n d / o r solvent controls. Variation between aliquots was typically less than 5% of the mean. For selected treatments, total ATP was divided by viable cell number to give the average ATP per cell.
Colony forming efficieno,. Two methods were used to determine colony-forming efficiency. In the 'replate' method, cells were washed at the end of the 3-h treatment, trypsinized, counted (Coulter counts) and plated at 100 cells/60-mm dish. In the 'in situ' method, cultures were seeded at 100 cells/60-mm dish, and 3 h later were treated, then washed after a further 3 h. All dishes (5 per treatment) from both methods were incubated in complete McCoy's 5A medium for 7 - 8 days, then colonies were stained with 0.4% methylene blue and counted. Results were expressed as a percentage of controls and the concentration which gave 50% relative survival (ECs0) was estimated from the d o s e - r e s p o n s e curve.
Acerage generation time estimates'. After the 3-h chemical treatment, cells were washed and allowed to recover for 26 h in complete McCoy's 5A medium supplemented with 5-bromodeoxyuridine at 10 /zM (BrdUrd; Sigma). About 3 h before harvest, colcemid (1(1 /xg/ml; G i b c o ) w a s added and accumulated mitotic cells were later collected by shakeoff, treated with hypotonic KCI (0.075 M) and fixed in 3 : 1 methanol : glacial acetic acid. Air-dried chromosome preparations were stained by the fluorescence-plus-Giemsa technique of Perry and Wolff (1974) modified by Goto et al. (1978). To estimate the average generation time (AGT), a replicative index (RI) based on 200 cells was calculated from the proportions of cells that had completed 1 (M1), 2 (M2) or between 1 and 2 (M1 + ) cycles in BrdUrd, RI = (M1 × 1 ) + ( M I + × 1 . 5 ) + ( M 2 × 2 ) . A G T (h) was calculated as the number of h in BrdUrd divided by the RI, and represents a rough estimate of the cell cycle time since all cells that had completed between 1 and 2 cycles were designated M1 + and treated as 1.5 cell cycles in the calculations.
48 TABLE 1 COMPARISON OF IN V I T R O M E T H O D S TO M E A S U R E T O X I C I T Y Treatments " dose
3hb
3hc
10 h
Counts
ATP
Counts
Counts
ADR (~M)
0.10 0.50 0.75 1.00
95 106 93 102
93 101 93 102
102 100 103 98
2-ABP (mM)
0.7 0.8 0.9 1.0 1.1 1.2
92 92 93 73 76 63
72 69 69 65 55 45
CdSO 4 (~M)
0.33 1.67 3.33
99 90 88
2,4-DAT (mM)
2 4 6 8
2,6-DAT (mM)
8 10 14 18
EUG (mM)
8-HQ (~M)
MMC (#M)
0.4 0.8 1.2 1.6 2.0 30 40 50 60 70 0.5 1.0 2.0 4.0 8.0
10 h
24-29 h
ATP
Counts
24-29 h ATP
MId
MI J
88 71 71 68
100 102 98 91
91 66 55 38
101 91 91 87
91 25 8 1
117 114 78 89
104 140 156 177
110 47 4 1
ND ND ND ND ND ND
96 77 84 66 59 8
94 92 84 76 55 9
94 79 57 57 28 8
82 75 72 61 33 8
97 57 64 49 4 3
97 88 67 69 63 14
106 112 133 151 183 188
72 64 83 58 23 2
102 85 77
ND ND ND
87 77 72
102 93 71
98 49 22
97 60 35
97 2 2
144 94 27
98 160 173
96 56 12
94 91 103 83
94 92 96 74
90 91 91 94
92 80 88 101
97 93 95 98
88 60 65 51
104 95 92 99
153 74 30 16
143 151 152 110
102 102 108 118
110 71 80 59
101 98 98 97
124 123 108 113
98 88 96 89
83 77 79 88
116 112 102 104
77 82 79 67
96 93 96 89
125 143 117 56
107 146 144 147
102 103 107 109
105 117 104 81
90 83 92 59 3
99 98 89 42 3
80 95 94 ,80 43
83 73 75 64 5
88 80 78 65 5
61 66 66 37 3
85 83 84 57 4
65 79 76 19 0
106 130 131 108 0
106 112 111 128 ND
69 114 76 59 0
99 46 22 13 6
100 45 24 14 5
94 97 84 73 62
89 71 59 26 18
94 78 61 30 17
96 48 32 15 9
102 77 53 23 15
100 1 3 1 1
92 42 21 19 13
101 114 110 107 107
65 25 18 19 5
109 104 96 105 ND
113 112 106 119 ND
74 83 78 73 67
99 89 99 97 86
105 111 110 100 107
96 69 57 67 51
94 96 101 90 90
34 33 26 25 11
100 153 95 61 25
122 176 191 > 194 > 194
89 48 10 2 0
AGT e
CFE f
Values are percentages of solvent, or combined solvent and negative controls. Viable cell number was assessed using trypan dye exclusion, except C3-h counts. a 3-h treatment. b These 3-h data for counts and ATP are from a separate experiment. All other data are from cultures treated concurrently. c Coulter cell counts. d Mitotic index. e Cell-cycle inhibition: A G T as % increase over control, control AGT ranged from 12.9 to 14.8 h. f Colony-forming efficiency: in-situ (2-ABP, CdSO 4) or replate data. ND, not determined concurrently with the other endpoints.
49
Mitotic index. At 10 and 24 or 29 h after the beginning of treatment all cells from colcemidtreated flasks were collected by trypsin harvest, fixed and stained with Giemsa. The mitotic index (MI) was the percentage of metaphase figures in 1000 cells. Chromosomal-aberration analysis. The A G T for C H O cells is 13-14 h. Colcemid arrested mitotic cells were harvested 10 and 24 h after the beginning of treatment. These cultures did not contain BrdUrd. Air-dried chromosome preparations were stained with Giemsa and 100-200 cells per treatment were scored for abs by one observer. Fewer than 200 cells were scored per treatment when ab frequencies were high or few cells were available for analysis due to mitotic suppression. Gaps, polyploid and endoreduplicated cells, and pulverized chromosomes were noted but not included in the ab totals. We classified as 'pulverized' chromatin that resembled prematurely condensed chromosomes from S-phase cells (Gollin et al., 1984). A cell with 10 or more abs was categorized as severely damaged and scored as 1 aberrant cell, but as 10 abs when calculating the total number of abs. Gaps were defined as achromatic lesions equal to or smaller than the width of the chromatid, and larger lesions with visible connecting material across the gap. Results are shown as the percentage of cells with abs (aberrant cells) and frequencies of abs per 100 cells. Statistical analysis of the percentages of cells with abs in treated cells compared with concurrent controls was by the 'normal test' of Margolin et al. (1983), a chi-squared test based on a standard normal approximation. Results
Details of toxicity and ab results are discussed for each compound below. Representative data comparing results of the toxicity endpoints are compiled in Table 1.
Adriamycin (Fig. 1) No toxicity as measured by cell counts was seen immediately after treatment for any dose between 0.1 and 2.0 /zM. A slight reduction in
24-
A
- ~ - t0HrMI
20 - % %
gv
12
28
-O-~ AGT24 Hr MI
24 if) -..q
d
20 "~
O c
8
-16 v
40100-
12 B
1
_~ -~ 80-
114
°t
I
E¢~ 60- ~ t~ .~ 40-
10 Hr 24Hr !
O-
120-
-
-
C
100 80
o~ 6o "6 ~ 40 20-
-~~ t0 HrCounts 24 Hr Counts ~
\
-
CFE
~m 0
II
0
~
~
0.10 0.25
~
0.50 ADR (pM)
~
0.75
1.00
Fig. 1. Cytotoxicity and chromosome aberration induction by adriamycin after 3-h treatment without $9: (A) MI at 10 and 24 h and AGT; (B) structural aberrations at 10 and 24 h, shown as % aberrant cells (% ab cells). The numbers above bars are frequencies of aberrations per 100 cells (shown when markedly different from % ab cells). % ab cells at all doses were significantly greater than combined controls, p < 0.05; (C) cell counts at 3, 10 and 24 h and CFE (replate).
cell number was observed at 10 h with doses between 0.1 and 1.0/zM while the CFE suppression was more than 50% at 0.5 tzM and above. Aberrations were induced by A D R at very low concentrations (17.5% aberrant cells at 0.1 /zM) in the absence of any acute or delayed toxicity. At 1.0 /zM, the few mitotic cells seen at 10 h had severe chromosome damage. More abs were seen at 10 than at 24 h at all concentrations of A D R . At low concentrations there was a lack of mitotic suppression or of change in A G T and as might be expected there was a higher ab yield at the earlier
50 h a r v e s t time. B u t at h i g h e r c o n c e n t r a t i o n s s u c h as 0 . 5 / z M , A D R was u n u s u a l in t h a t e v e n w h e n t h e M I a n d A G T i n d i c a t e d cell-cycle d e l a y t h e yield o f abs was h i g h e r at 10 t h a n 24 h a l t h o u g h it m i g h t be e x p e c t e d t h a t d e l a y e d cells w o u l d app e a r at 24 h. T h e d e c r e a s e in abs w i t h t i m e c o u l d be d u e to d e a t h o r a r r e s t o f cells w i t h abs, b u t if so, o n l y a small p r o p o r t i o n o f t h e cell p o p u l a t i o n is a f f e c t e d since t h e i n c r e a s e in a b s o l u t e cell n u m b e r f r o m 10 to 24 h s h o w e d t h a t at 0.5 # M cells w e r e g r o w i n g as fast as c o n t r o l s ( d a t a n o t
20-
:s
-
24
12 - 20
N
a
"7O E
-16 ~
4 0 25
> O
Q) ..D
< o4
10
/,'
I
I
I
I
12
~
27!
B
17 ~
i
24Hr
16~ 12
B
444
~o 8O 60
10 Hr 24 Hr
40
118
O4 2O 0 100 80-
"5 O4 40-
10
|
1
~
-~- 10HrCounts - I - 29 HrCounts - ¢ ~ ~ ~
0 ----F-/ 0 0.7
~
~
0.8 0.9 1.0 2-ABP (raM)
~. \ ~ \ 1 ~ 1.1
3 Hr Counts
~ -0-
0 Hr C
o
u
n
29 Hr C
o
u
n
I I
1.2
Fig. 2. Cytotoxicity and chromosome aberration induction by 2-aminobiphenyl after 3-h treatment with $9: (A) MI at 10 and 29 h and AGT; (B) structural aberrations at 10 and 24 h, shown as % aberrant cells (% ab cells). The numbers above bars are frequencies of aberrations per 100 cells (shown when markedly different from % ab cells). %. ab cells at 1.1 and 1.2 mM at 24 h were significantly greater than DMSO control, P < 0.05; (C) cell counts at 3, 10 and 29 h and CFE (in situ). 3-h counts were from a separate experiment.
t
s t
~ s
~
CFE
~
0 0.3
60 O4 20
o E
~1
10 Hr
0 - ~ ¢ ~---t----100 8O C ~
,.6 40
8-
20-
5
o
--4
20 "~
60I
20 0 15 Erd
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0 100-
43
