Mutation Research, 265 (1992) 31-44 (c~ 1992 Elsevier Science Publishers B.V. All rights reserved 11027-5107/92/$1)5.011
31
MUT 05032
Effect of sampling time on chromosome aberration yield for 7 chemicals in Chinese hamster ovary cells Christian L. Bean, Michael J. Armstrong and Sheila M. Galloway Merck Sharp and Dohme Research Lahoratorie~, W45-305, West Point, PA 19486 (U.S.A.) (Received 1 February 1991) (Revision received 11 April t991) (Accepted I July 1991)
Keywords: Chromosome aberrations: CLIO, in vitro: Kinetics: Sampling time: Clastogcns: Endorcduplication
Summary Choice of harvest time is one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. We examined the effects of sampling time on aberration yield for 7 diverse chemicals in CHO-WBL cells by harvesting at intervals from 9 to 3(I h after treatment for 3 h with or without $9 metabolic activation. We observed both the percentage of aberrant cells and the total number of aberrations. Our data suggest that for most compounds a single harvest time approximately 17-21 h after the beginning of a 3-h treatment is optimal for aberration detection in CHO cells. Maximal aberration yields were observed for 2,4-diaminotoluene, 2,6-diaminotoluene and cytosine /3-D-arabinofuranoside from 17 to 21 h, eugenol from 15 to 21 h, cadmium sulfate from 15 to 24 h and 2-aminobiphenyl, from 17 to 24 h. For adriamycin at 1 /zU, the % aberrant cells remained elevated throughout the period from 9 to 29 h, while small increases at 0.1 /~M A D R were found only at 13 and at 25 h. For most chemicals the maximal aberration yield occurred at a different time for each concentration tested. However, the use of 3 or more closely spaced concentrations, carefully selected to yield up to 50% toxicity, allowed detection of a positive response at a single harvest time for all 7 chemicals.
The in vitro chromosome aberration assay in Chinese hamster ovary (CHO) cells is widely used to test chemicals for their clastogenic potential (e.g., Galloway et al., 1985, 1987a). In efforts to optimize a n d / o r standardize the assays, many factors have been studied including exposure time,
Correspondence: Dr. Sheila M. Galloway, Merck Sharp and Dohme Research Laboratories, WP45-3(J4, West Point, PA 19486 (U.S.A.).
maximum limits on concentration, cytotoxicity and osmolality, and effects of the $9 metabolic activation system and of pH (Armstrong et al., 1992; Galloway et al., 1987b, 1990; Brusick, 1986; Morita et al., 1989, 1990). However, only a few studies exist on selection of harvest time(s) for maximal aberration yields after chemical exposure (Nowak, 1990; Ceccherini, 1988; Parkes and Scott, 1982; Thust et al., 1980; Thust, 1982; Rohr and Bauchinger, 1976). Thust 11982) found that the time course of aberration yields may differ
32 widely even among closely related compounds. Choosing the correct harvest time is vitally important to optimize the yield of aberrations. Since aberrations induced by most chemicals are produced during DNA replication, harvest time must allow cells to progress through S-phase after treatment. It is ideal to score aberrations in the first metaphase after they are formed to avoid loss during mitosis or conversion of the initial aberrations into complex derivatives during subsequent cell cycles. 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 in CHO cells which have a cell-cycle length of 12-14 h (Galloway et al., 1985). However, dose-dependent cell-cycle delay may result from clastogen exposure, so that to detect aberrations delayed cells must be allowed sufficient time to progress to mitosis and sampling times of, e.g., 18-28 h have proved appropriate in CHO cells (e.g., Galloway et al., 1987a; Loveday et al., 1989). Choice of harvest time is critical to detection of weakly active chemicals. In addition, evidence for a dose relation may be missed at an inappropriate harvest time, e.g., a detectable aberration response for a higher dose may occur later than that for a lower dose due to greater cell-cycle delay, so at a given harvest time the response might be found at only one dose. Here we examined chromosome aberration kinetics for 7 chemicals by harvesting at intervals from 9 to 30 h from the beginning of the 3-h treatment. The chemicals are a diverse group with different modes of action, and include rodent carcinogens, weak carcinogens and noncarcinogens (Ashby and Tennant, 1988; Heath et al., 1962; Tennant et al., 1987; Marquardt et al., 1976). They all inhibit cell-cycle progression, although with varying efficiencies (Bean, Selden, Miller and Galloway, unpublished data). We examined both the percentage of cells with structural aberrations (% ab cells) and the total number of aberrations per 100 cells (total abs). Materials and methods
Culture of CHO WBL cells CHO cells were obtained in 1979 from Dr. S. Wolff, University of California at San Francisco,
and have since been cloned repeatedly. The cells were maintained in complete medium, i.e., McCoy's 5A medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Hazleton, Lenexa~ KS or 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. Cells were routinely subcultured at ratios of 1-10 or 1-20. Cells were not used after the 15th passage since cloning, and were generally between population doublings 60 and 90.
Metabolic actit~ation system Liver homogenate ($9 fraction) was prepared from phenobarbital//3-naphthoflavone induced male CRCD Crl:CD(SD)BR Sprague-Dawley rats from Charles River Breeding Farms, Raleigh, NC. $9 was stored at - 7 0 ° C to - 8 0 °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 were: $9, 15 ~l / m l ; NADP, 0.8 mg/ml (1.05 mM) and trisodium isocitrate, 1.5 mg/ml (5.8 mM). Assessment of chromosome aberration kinetics About 24 h before treatment, 1.2 × 106 ceils were seeded in 10 ml medium in 75 cm: 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 2+ and Mg 2+ (DPBS, Gibco) and refed with 9.9 ml of serumfree medium containing the $9 metabolic activation system. Test compound (100 ~1) was added from concentrated stocks (100 ×), and cultures both with and without $9 mix were incubated for 3 h at 37°C in a humidified, 5% CO 2 atmosphere, then washed twice with warmed DPBS and refed with 10 ml of prewarmed complete medium. Flasks were harvested at intervals between 9 and 30 h from the beginning of treatment, and 2.5-3 h after addition of 0.1 /zg/ml colcemid (Gibco). Ceils were harvested by mitotic shake-off,
33 or by trypsinization so that samples could be taken to count cells in a Coulter counter (model ZM, Coulter Electronics, Hialeah, FL). Harvested cells were then treated with hypotonic solution (KC1 75 mM) for 1-3 min at room temperature, washed twice with fixative (methanol: glacial acetic acid, 3 : 1 v/v), dropped onto slides, air-dried, stained with 5% Giemsa then mounted.
Test chemicals and solvents Adriamycin (ADR; doxorubicin hydrochloride, CAS No. 97-53-0), 2-aminobiphenyl (2-ABP; 2-biphenylamine, CAS No. 90-41-5), cadmium sulfate (CdSO4; CAS No. 10124-36-4), cytosine /3-oarabinofuranoside (Ara-C; CAS No. 147-94-4), and dimethyl sulfoxide (DMSO) were from Sigma, St. Louis, MO. 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. Deionized, distilled water (dHzO) was purchased from Gibco. ADR, CdSO 4 and Ara-C were prepared in dH20. 2ABP, 2,4-DAT, 2,6-DAT and EUG were prepared in DMSO. EUG and 2-ABP were tested in the presence of $9 metabolic activation. None changed the pH of the medium as judged by the color of the indicator. Osmolality was measured in a vapor pressure osmometer (model 5500, Wescor, Logan, UT). Toxicity assessment Toxicity was assessed by estimates of monolayer confluence and observations of abnormal cell morphology, or by Coulter cell counts of trypsinized cultures (see above). Monolayer confluence was determined using a light microscope by observing the extent of cell proliferation on the growth surface compared with concurrent controls. Estimates of confluence were within 10% of Coulter cell counts when both measurements were made. Dose selection The maximum concentrations to be tested were based on previous experiments and generally did not exceed 50% reductions of cell counts compared with concurrent controls. The maximum concentration tested was less than 10 mM except for 2,6-DAT which was tested up to 18 mM due
to lack of cytotoxicity, but did not increase the osmolality of the test medium over that of the 1% DMSO control.
Aberration scoring Where possible, 200 cells per point were scored for aberrations unless there were >/50% aberrant cells, when 50 cells were scored. At four points with >/30% aberrant cells, 100 cells were scored. Only intact cells with good chromosome morphology and having no overlap with other nuclei or debris were scored. We did not score aberrations in polyploid cells, and used metaphases with 19-23 chromosomes, the modal number being 21. All types of structural aberrations were recorded. Chromatid and isochromatid gaps were recorded but not included in the totals of aberrations. 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. Polyploid and endoreduplicated cells were also noted but were not included in aberration totals. We calculated both the percentage of aberrant cells (% ab cells, the number of cells with structural aberration(s) per 100 cells) and the frequency of aberrations (total abs, the total number of aberrations per 100 cells), since a cell may have more than one aberration. Cells with 10 or more aberrations were classed as severely damaged (SD) cells, and scored as one aberrant cell but as 10 aberrations. 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. Results
Adriamycin (Fig. 1). The higher dose of ADR scored for aberrations (1.0 ~M) did not cause any appreciable reduction in cell numbers (Coulter counts) relative to controls, from 9 to 29 h after dosing. At 1.0 /~M, we observed a dramatic increase in both the % ab cells (74.0%) and total abs (198/100 cells) at the earliest observation time, 9 h (Fig. 1). (Note that the scale for total abs in Fig. 1 was adjusted to allow for the high
34 P e
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I t0
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200
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:
/
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25
30
of treatment)
Fig. 1. Chromosome aberration induction by 1.0 /xM ADR without $9 at various times after the beginning of a 3-h treatment in CHO cells. 50 cells scored per point. - - , the percentage of aberrant cells (% ab cells), closed symbols; . . . . . . , the frequency of aberrations per 100 cells, open symbols. The mean % ab cells (and range across times) for controls was 1.9% (1.0-3.0%). Increases with ADR were significant (P < 0.001) for all times.
incidence of aberrations.) Both measurements increased to a peak at 21 h and were still very high at 29 h. Predominantly chromatid breaks and interchanges were seen at 9 and 13 h, while at 17 h and later some dicentrics and rings (14-26 per 100 cells) were also found. Isochromatid deletions were observed at 9 h (34/100 cells) and were very numerous by 21 h (182/100 cells) through 29 h. By 29 h there were no chromatid interchanges. The number of SD cells was maximal at 21 h and fell to 0 by 25 h. Many endoreduplicated cells (13.8% of 500 metaphases) were observed at 21 h, which was the same time as the peak for structural aberrations. Increased numbers of endoreduplicated cells (2.4%) were seen at only one other time, 25 h. Controls had 0.2%. Thus, at 1.0 /~M A D R aberrations were detected at all the harvest times, with maximum yields at 17 and 21 h. However, at the lower dose (0.1 /xM) small but significant increases were detected only at 13 and at 25 h
(7% ab cells at each time; P ~< 0.05 cf. controls of 1.5-3.0%). At other times the % ab cells never exceeded 5.0%.
2-Aminobiphenyl (with S9) (Figs. 2a,b and 3). 2-ABP induced aberrations at rather cytotoxic doses (0.9-1.2 raM). The highest dose exceeded our usual limit of toxicity of 50%, since 1.2 mM 2-ABP gave 41 and 22% of control cell number (Coulter counts) at 15 and 25 h respectively. Cell numbers at 15 and 25 h were 55 and 42% of controls at 1.1 mM 2-ABP, and 65 and 70% of controls at 1.0 mM 2-ABP. For 2-ABP the maximum % ab cells shifted to later times with increasing dose (Fig. 2a, 0.9 mM: 9.5% at 17.0 h; 1.0 raM: 6.0% at 19.5 h; 1.1 mM: 19.5% at 22.0 h and 1.2 mM: 18.5% at 22.0 h; P ~< 0.02 for all determinations). The total abs followed the same pattern (data not shown) except where 1 SD cell (cell with 10 or more aberrations) at each of 0.9 and 1.0 mM inflated the totals at 24 h. In a repeat experiment (Fig. 2b), the maximum yields of % ab cells and of total abs were at slightly later times than in the first experiment (Fig. 2a), e.g., at 0.9 mM the maximal yields both of % ab cells (10%) and of total abs (18/100) were at 19.5 h. The shift of the peaks to later times in the second experiment is consistent with the slightly greater toxicity in this experiment, seen from monolayer confluence and mitotic suppression seen in the cultures before harvest. Chromatid and isochromatid deletions and chromatid exchanges were observed at each dose of 2-ABP but there was no increase in chromosome exchanges. At 1.0-1.2 mM 2-ABP, isochromatid gaps and chromosome breaks were observed in the secondary constriction area of the long arm of the X chromosome (Xq). This region is often affected when highly toxic conditions are encountered. The most distal portion of the long arm was elongated, resembling a large gap or isochromatid gap with visible fine connecting material. Breaks in this region were scored only when there was clearly no connecting material in the achromatic region or when there was clear displacement of the chromatid(s). Breaks in Xq accounted for up to one third of the aberrant cells, e.g., at 1.2 mM at 24.5 h in the second experiment.
35
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Fig. 2. Chromosome aberration induction by 2-ABP with $9 at various times after a 3-h treatment in C H O cells. 200 cells scored per point except at 1.2 m M at 15 h in Expt. 2(b) where 186 cells were scored. The mean % ab cells (and range across times) for controls were: 1.6% ( 1 - 2 % ) for Expt. l(a) and 2.5% (1.0-3.5%) for Expt. 2(b). Increases were significant ( P < 0.05) for 0.9 mM at 17.0 h, 1.0 m M at 19.5 h, 1.1 and 1.2 m M at 17.0, 19.5, 22.0, and 24.5 h; and for Expt. 2(b) for 0.9 m M at 19.5 h, 1.0 at 19.5 and 22.0 h, 1.1 mM at 19.5, 22.0 and 24.5 h and 1.2 m M at 22.0 and 24.5 h.
Many endoreduplicated cells were noted in both experiments, usually with maximum frequencies 2.5-3.0 h later than those for structural aberrations (Fig. 3, data shown for one experiment). ENDOREDUPLICATION 2-ABP
%
6
0
0.8 mM 0.9 mM 1.0 mM
)mLi[, 15.0
17.0 J.9.5 22.0 Hrs tfrom start of treatment}
245
Fig. 3. Percentage of endoreduplicated cells in 500 mitoses. C H O cells were exposed to 2-ABP with $9 for 3 h and were harvested 15.0-24.5 h from the beginning of treatment.
The data for 2-ABP illustrate the very narrow dose range and degree of cytotoxicity needed to detect aberrations. At 19.5 h, statistically significant increases (P ~
31
MUT 05032
Effect of sampling time on chromosome aberration yield for 7 chemicals in Chinese hamster ovary cells Christian L. Bean, Michael J. Armstrong and Sheila M. Galloway Merck Sharp and Dohme Research Lahoratorie~, W45-305, West Point, PA 19486 (U.S.A.) (Received 1 February 1991) (Revision received 11 April t991) (Accepted I July 1991)
Keywords: Chromosome aberrations: CLIO, in vitro: Kinetics: Sampling time: Clastogcns: Endorcduplication
Summary Choice of harvest time is one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. We examined the effects of sampling time on aberration yield for 7 diverse chemicals in CHO-WBL cells by harvesting at intervals from 9 to 3(I h after treatment for 3 h with or without $9 metabolic activation. We observed both the percentage of aberrant cells and the total number of aberrations. Our data suggest that for most compounds a single harvest time approximately 17-21 h after the beginning of a 3-h treatment is optimal for aberration detection in CHO cells. Maximal aberration yields were observed for 2,4-diaminotoluene, 2,6-diaminotoluene and cytosine /3-D-arabinofuranoside from 17 to 21 h, eugenol from 15 to 21 h, cadmium sulfate from 15 to 24 h and 2-aminobiphenyl, from 17 to 24 h. For adriamycin at 1 /zU, the % aberrant cells remained elevated throughout the period from 9 to 29 h, while small increases at 0.1 /~M A D R were found only at 13 and at 25 h. For most chemicals the maximal aberration yield occurred at a different time for each concentration tested. However, the use of 3 or more closely spaced concentrations, carefully selected to yield up to 50% toxicity, allowed detection of a positive response at a single harvest time for all 7 chemicals.
The in vitro chromosome aberration assay in Chinese hamster ovary (CHO) cells is widely used to test chemicals for their clastogenic potential (e.g., Galloway et al., 1985, 1987a). In efforts to optimize a n d / o r standardize the assays, many factors have been studied including exposure time,
Correspondence: Dr. Sheila M. Galloway, Merck Sharp and Dohme Research Laboratories, WP45-3(J4, West Point, PA 19486 (U.S.A.).
maximum limits on concentration, cytotoxicity and osmolality, and effects of the $9 metabolic activation system and of pH (Armstrong et al., 1992; Galloway et al., 1987b, 1990; Brusick, 1986; Morita et al., 1989, 1990). However, only a few studies exist on selection of harvest time(s) for maximal aberration yields after chemical exposure (Nowak, 1990; Ceccherini, 1988; Parkes and Scott, 1982; Thust et al., 1980; Thust, 1982; Rohr and Bauchinger, 1976). Thust 11982) found that the time course of aberration yields may differ
32 widely even among closely related compounds. Choosing the correct harvest time is vitally important to optimize the yield of aberrations. Since aberrations induced by most chemicals are produced during DNA replication, harvest time must allow cells to progress through S-phase after treatment. It is ideal to score aberrations in the first metaphase after they are formed to avoid loss during mitosis or conversion of the initial aberrations into complex derivatives during subsequent cell cycles. 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 in CHO cells which have a cell-cycle length of 12-14 h (Galloway et al., 1985). However, dose-dependent cell-cycle delay may result from clastogen exposure, so that to detect aberrations delayed cells must be allowed sufficient time to progress to mitosis and sampling times of, e.g., 18-28 h have proved appropriate in CHO cells (e.g., Galloway et al., 1987a; Loveday et al., 1989). Choice of harvest time is critical to detection of weakly active chemicals. In addition, evidence for a dose relation may be missed at an inappropriate harvest time, e.g., a detectable aberration response for a higher dose may occur later than that for a lower dose due to greater cell-cycle delay, so at a given harvest time the response might be found at only one dose. Here we examined chromosome aberration kinetics for 7 chemicals by harvesting at intervals from 9 to 30 h from the beginning of the 3-h treatment. The chemicals are a diverse group with different modes of action, and include rodent carcinogens, weak carcinogens and noncarcinogens (Ashby and Tennant, 1988; Heath et al., 1962; Tennant et al., 1987; Marquardt et al., 1976). They all inhibit cell-cycle progression, although with varying efficiencies (Bean, Selden, Miller and Galloway, unpublished data). We examined both the percentage of cells with structural aberrations (% ab cells) and the total number of aberrations per 100 cells (total abs). Materials and methods
Culture of CHO WBL cells CHO cells were obtained in 1979 from Dr. S. Wolff, University of California at San Francisco,
and have since been cloned repeatedly. The cells were maintained in complete medium, i.e., McCoy's 5A medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Hazleton, Lenexa~ KS or 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. Cells were routinely subcultured at ratios of 1-10 or 1-20. Cells were not used after the 15th passage since cloning, and were generally between population doublings 60 and 90.
Metabolic actit~ation system Liver homogenate ($9 fraction) was prepared from phenobarbital//3-naphthoflavone induced male CRCD Crl:CD(SD)BR Sprague-Dawley rats from Charles River Breeding Farms, Raleigh, NC. $9 was stored at - 7 0 ° C to - 8 0 °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 were: $9, 15 ~l / m l ; NADP, 0.8 mg/ml (1.05 mM) and trisodium isocitrate, 1.5 mg/ml (5.8 mM). Assessment of chromosome aberration kinetics About 24 h before treatment, 1.2 × 106 ceils were seeded in 10 ml medium in 75 cm: 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 2+ and Mg 2+ (DPBS, Gibco) and refed with 9.9 ml of serumfree medium containing the $9 metabolic activation system. Test compound (100 ~1) was added from concentrated stocks (100 ×), and cultures both with and without $9 mix were incubated for 3 h at 37°C in a humidified, 5% CO 2 atmosphere, then washed twice with warmed DPBS and refed with 10 ml of prewarmed complete medium. Flasks were harvested at intervals between 9 and 30 h from the beginning of treatment, and 2.5-3 h after addition of 0.1 /zg/ml colcemid (Gibco). Ceils were harvested by mitotic shake-off,
33 or by trypsinization so that samples could be taken to count cells in a Coulter counter (model ZM, Coulter Electronics, Hialeah, FL). Harvested cells were then treated with hypotonic solution (KC1 75 mM) for 1-3 min at room temperature, washed twice with fixative (methanol: glacial acetic acid, 3 : 1 v/v), dropped onto slides, air-dried, stained with 5% Giemsa then mounted.
Test chemicals and solvents Adriamycin (ADR; doxorubicin hydrochloride, CAS No. 97-53-0), 2-aminobiphenyl (2-ABP; 2-biphenylamine, CAS No. 90-41-5), cadmium sulfate (CdSO4; CAS No. 10124-36-4), cytosine /3-oarabinofuranoside (Ara-C; CAS No. 147-94-4), and dimethyl sulfoxide (DMSO) were from Sigma, St. Louis, MO. 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. Deionized, distilled water (dHzO) was purchased from Gibco. ADR, CdSO 4 and Ara-C were prepared in dH20. 2ABP, 2,4-DAT, 2,6-DAT and EUG were prepared in DMSO. EUG and 2-ABP were tested in the presence of $9 metabolic activation. None changed the pH of the medium as judged by the color of the indicator. Osmolality was measured in a vapor pressure osmometer (model 5500, Wescor, Logan, UT). Toxicity assessment Toxicity was assessed by estimates of monolayer confluence and observations of abnormal cell morphology, or by Coulter cell counts of trypsinized cultures (see above). Monolayer confluence was determined using a light microscope by observing the extent of cell proliferation on the growth surface compared with concurrent controls. Estimates of confluence were within 10% of Coulter cell counts when both measurements were made. Dose selection The maximum concentrations to be tested were based on previous experiments and generally did not exceed 50% reductions of cell counts compared with concurrent controls. The maximum concentration tested was less than 10 mM except for 2,6-DAT which was tested up to 18 mM due
to lack of cytotoxicity, but did not increase the osmolality of the test medium over that of the 1% DMSO control.
Aberration scoring Where possible, 200 cells per point were scored for aberrations unless there were >/50% aberrant cells, when 50 cells were scored. At four points with >/30% aberrant cells, 100 cells were scored. Only intact cells with good chromosome morphology and having no overlap with other nuclei or debris were scored. We did not score aberrations in polyploid cells, and used metaphases with 19-23 chromosomes, the modal number being 21. All types of structural aberrations were recorded. Chromatid and isochromatid gaps were recorded but not included in the totals of aberrations. 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. Polyploid and endoreduplicated cells were also noted but were not included in aberration totals. We calculated both the percentage of aberrant cells (% ab cells, the number of cells with structural aberration(s) per 100 cells) and the frequency of aberrations (total abs, the total number of aberrations per 100 cells), since a cell may have more than one aberration. Cells with 10 or more aberrations were classed as severely damaged (SD) cells, and scored as one aberrant cell but as 10 aberrations. 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. Results
Adriamycin (Fig. 1). The higher dose of ADR scored for aberrations (1.0 ~M) did not cause any appreciable reduction in cell numbers (Coulter counts) relative to controls, from 9 to 29 h after dosing. At 1.0 /~M, we observed a dramatic increase in both the % ab cells (74.0%) and total abs (198/100 cells) at the earliest observation time, 9 h (Fig. 1). (Note that the scale for total abs in Fig. 1 was adjusted to allow for the high
34 P e
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e
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e
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75" ,400
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e
r
50-
G
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/ ;;
n
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t
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'".%
I t0
Hrs
200
"d
25-
0
:
/
I t5
(from s t a r t
!
I
20
25
30
of treatment)
Fig. 1. Chromosome aberration induction by 1.0 /xM ADR without $9 at various times after the beginning of a 3-h treatment in CHO cells. 50 cells scored per point. - - , the percentage of aberrant cells (% ab cells), closed symbols; . . . . . . , the frequency of aberrations per 100 cells, open symbols. The mean % ab cells (and range across times) for controls was 1.9% (1.0-3.0%). Increases with ADR were significant (P < 0.001) for all times.
incidence of aberrations.) Both measurements increased to a peak at 21 h and were still very high at 29 h. Predominantly chromatid breaks and interchanges were seen at 9 and 13 h, while at 17 h and later some dicentrics and rings (14-26 per 100 cells) were also found. Isochromatid deletions were observed at 9 h (34/100 cells) and were very numerous by 21 h (182/100 cells) through 29 h. By 29 h there were no chromatid interchanges. The number of SD cells was maximal at 21 h and fell to 0 by 25 h. Many endoreduplicated cells (13.8% of 500 metaphases) were observed at 21 h, which was the same time as the peak for structural aberrations. Increased numbers of endoreduplicated cells (2.4%) were seen at only one other time, 25 h. Controls had 0.2%. Thus, at 1.0 /~M A D R aberrations were detected at all the harvest times, with maximum yields at 17 and 21 h. However, at the lower dose (0.1 /xM) small but significant increases were detected only at 13 and at 25 h
(7% ab cells at each time; P ~< 0.05 cf. controls of 1.5-3.0%). At other times the % ab cells never exceeded 5.0%.
2-Aminobiphenyl (with S9) (Figs. 2a,b and 3). 2-ABP induced aberrations at rather cytotoxic doses (0.9-1.2 raM). The highest dose exceeded our usual limit of toxicity of 50%, since 1.2 mM 2-ABP gave 41 and 22% of control cell number (Coulter counts) at 15 and 25 h respectively. Cell numbers at 15 and 25 h were 55 and 42% of controls at 1.1 mM 2-ABP, and 65 and 70% of controls at 1.0 mM 2-ABP. For 2-ABP the maximum % ab cells shifted to later times with increasing dose (Fig. 2a, 0.9 mM: 9.5% at 17.0 h; 1.0 raM: 6.0% at 19.5 h; 1.1 mM: 19.5% at 22.0 h and 1.2 mM: 18.5% at 22.0 h; P ~< 0.02 for all determinations). The total abs followed the same pattern (data not shown) except where 1 SD cell (cell with 10 or more aberrations) at each of 0.9 and 1.0 mM inflated the totals at 24 h. In a repeat experiment (Fig. 2b), the maximum yields of % ab cells and of total abs were at slightly later times than in the first experiment (Fig. 2a), e.g., at 0.9 mM the maximal yields both of % ab cells (10%) and of total abs (18/100) were at 19.5 h. The shift of the peaks to later times in the second experiment is consistent with the slightly greater toxicity in this experiment, seen from monolayer confluence and mitotic suppression seen in the cultures before harvest. Chromatid and isochromatid deletions and chromatid exchanges were observed at each dose of 2-ABP but there was no increase in chromosome exchanges. At 1.0-1.2 mM 2-ABP, isochromatid gaps and chromosome breaks were observed in the secondary constriction area of the long arm of the X chromosome (Xq). This region is often affected when highly toxic conditions are encountered. The most distal portion of the long arm was elongated, resembling a large gap or isochromatid gap with visible fine connecting material. Breaks in this region were scored only when there was clearly no connecting material in the achromatic region or when there was clear displacement of the chromatid(s). Breaks in Xq accounted for up to one third of the aberrant cells, e.g., at 1.2 mM at 24.5 h in the second experiment.
35
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Fig. 2. Chromosome aberration induction by 2-ABP with $9 at various times after a 3-h treatment in C H O cells. 200 cells scored per point except at 1.2 m M at 15 h in Expt. 2(b) where 186 cells were scored. The mean % ab cells (and range across times) for controls were: 1.6% ( 1 - 2 % ) for Expt. l(a) and 2.5% (1.0-3.5%) for Expt. 2(b). Increases were significant ( P < 0.05) for 0.9 mM at 17.0 h, 1.0 m M at 19.5 h, 1.1 and 1.2 m M at 17.0, 19.5, 22.0, and 24.5 h; and for Expt. 2(b) for 0.9 m M at 19.5 h, 1.0 at 19.5 and 22.0 h, 1.1 mM at 19.5, 22.0 and 24.5 h and 1.2 m M at 22.0 and 24.5 h.
Many endoreduplicated cells were noted in both experiments, usually with maximum frequencies 2.5-3.0 h later than those for structural aberrations (Fig. 3, data shown for one experiment). ENDOREDUPLICATION 2-ABP
%
6
0
0.8 mM 0.9 mM 1.0 mM
)mLi[, 15.0
17.0 J.9.5 22.0 Hrs tfrom start of treatment}
245
Fig. 3. Percentage of endoreduplicated cells in 500 mitoses. C H O cells were exposed to 2-ABP with $9 for 3 h and were harvested 15.0-24.5 h from the beginning of treatment.
The data for 2-ABP illustrate the very narrow dose range and degree of cytotoxicity needed to detect aberrations. At 19.5 h, statistically significant increases (P ~
